scholarly journals Marker-assisted mapping enables effective forward genetic analysis in the arboviral vector Aedes aegypti, a species with vast recombination deserts

2021 ◽  
Author(s):  
Chujia Chen ◽  
Austin Compton ◽  
Katerina Nikolouli ◽  
Aihua Wang ◽  
Azadeh Aryan ◽  
...  
Keyword(s):  
Genetic Analysis ◽  
Aedes Aegypti ◽  
Reverse Genetics ◽  
Signal To Noise Ratio ◽  
Forward Genetics ◽  
Genetic Identification ◽  
Vector Biology ◽  
Causal Gene ◽  
Genetic Sexing ◽  
Important Species

AbstractAedes aegypti is a major vector of arboviruses that cause dengue, chikungunya, yellow fever and Zika. Although recent success in reverse genetics has facilitated rapid progress in basic and applied research, integration of forward genetics with modern technologies remains challenging in this important species, as up-to-47% of its chromosome is refractory to genetic mapping due to extremely low rate of recombination. Here we report the development of a marker-assisted-mapping (MAM) strategy to readily screen for and genotype only the rare but informative recombinants, drastically increasing both the resolution and signal-to-noise ratio. Using MAM, we mapped a transgene that was inserted in a >100 Mb recombination desert and a sex-linked spontaneous red-eye (re) mutation just outside the region. We subsequently determined, by CRISPR/Cas9-mediated knockout, that cardinal is the causal gene of re, which is the first forward genetic identification of a causal gene in Ae. aegypti. This study provides the molecular foundation for using gene-editing to develop versatile and stable genetic sexing methods by improving upon the current re-based genetic sexing strains. MAM does not require densely populated markers and can be readily applied throughout the genome to facilitate the mapping of genes responsible for insecticide- and viral-resistance. By enabling effective forward genetic analysis, MAM bridges a significant gap in establishing Ae. aegypti as a model system for research in vector biology. As large regions of suppressed recombination are also common in other plant and animal species including those of economic significance, MAM will have broad applications beyond vector biology.

scholarly journals Review of Current Methodological Approaches for Characterizing MicroRNAs in Plants

2009 ◽  
Vol 2009 ◽  
pp. 1-11 ◽  
Author(s):  
Turgay Unver ◽  
Deana M. Namuth-Covert ◽  
Hikmet Budak
Keyword(s):  
Gene Expression ◽  
Molecular Biology ◽  
Reverse Genetics ◽  
Methodological Approach ◽  
Forward Genetics ◽  
Methodological Approaches

Advances in molecular biology have led to some surprising discoveries. One of these includes the complexities of RNA and its role in gene expression. One particular class of RNA called microRNA (miRNA) is the focus of this paper. We will first briefly look at some of the characteristics and biogenesis of miRNA in plant systems. The remainder of the paper will go into details of three different approaches used to identify and study miRNA. These include two reverse genetics approaches: computation (bioinformatics) and experimental, and one rare forward genetics approach. We also will summarize how to measure and quantify miRNAs, and how to detect their possible targets in plants. Strengths and weaknesses of each methodological approach are discussed.

scholarly journals Creating Porcine Biomedical Models Through Recombineering

2004 ◽  
Vol 5 (3) ◽  
pp. 262-267 ◽  
Author(s):  
Margarita M. Rogatcheva ◽  
Laurie A. Rund ◽  
Kelly S. Swanson ◽  
Brandy M. Marron ◽  
Jonathan E. Beever ◽  
...  
Keyword(s):  
Genetic Information ◽  
Positional Cloning ◽  
Reverse Genetics ◽  
Genetic Model ◽  
Phenotypic Diversity ◽  
Model Organism ◽  
Artificial Chromosome ◽  
Forward Genetics ◽  
Working Definition ◽  
Definition Of

Recent advances in genomics provide genetic information from humans and other mammals (mouse, rat, dog and primates) traditionally used as models as well as new candidates (pigs and cattle). In addition, linked enabling technologies, such as transgenesis and animal cloning, provide innovative ways to design and perform experiments to dissect complex biological systems. Exploitation of genomic information overcomes the traditional need to choose naturally occurring models. Thus, investigators can utilize emerging genomic knowledge and tools to create relevant animal models. This approach is referred to as reverse genetics. In contrast to ‘forward genetics’, in which gene(s) responsible for a particular phenotype are identified by positional cloning (phenotype to genotype), the ‘reverse genetics’ approach determines the function of a gene and predicts the phenotype of a cell, tissue, or organism (genotype to phenotype). The convergence of classical and reverse genetics, along with genomics, provides a working definition of a ‘genetic model’ organism (3). The recent construction of phenotypic maps defining quantitative trait loci (QTL) in various domesticated species provides insights into how allelic variations contribute to phenotypic diversity. Targeted chromosomal regions are characterized by the construction of bacterial artificial chromosome (BAC) contigs to isolate and characterize genes contributing towards phenotypic variation. Recombineering provides a powerful methodology to harvest genetic information responsible for phenotype. Linking recombineering with gene-targeted homologous recombination, coupled with nuclear transfer (NT) technology can provide ‘clones’ of genetically modified animals.

Phenotypic Screen and Transcriptomics Approach Complement Each Other in Functional Genomics of Defensive Stink Gland Physiology

2021 ◽  
Author(s):  
Sabrina Lehmann ◽  
Bibi Atika ◽  
Daniela Grossmann ◽  
Christian Schmitt-Engel ◽  
Nadi Strohlein ◽  
...  
Keyword(s):  
Functional Genomics ◽  
Large Scale ◽  
Reverse Genetics ◽  
Expression Profiles ◽  
Forward Genetics ◽  
Large Set ◽  
Knock Down ◽  
Genome Wide ◽  
Phenotypic Screen ◽  
A Genome

Abstract Background Functional genomics uses unbiased systematic genome-wide gene disruption or analyzes natural variations such as gene expression profiles of different tissues from multicellular organisms to link gene functions to particular phenotypes. Functional genomics approaches are of particular importance to identify large sets of genes that are specifically important for a particular biological process beyond known candidate genes, or when the process has not been studied with genetic methods before. Results Here, we present a large set of genes whose disruption interferes with the function of the odoriferous defensive stink glands of the red flour beetle Tribolium castaneum. This gene set is the result of a large-scale systematic phenotypic screen using a reverse genetics strategy based on RNA interference applied in a genome-wide forward genetics manner. In this first-pass screen, 130 genes were identified, of which 69 genes could be confirmed to cause knock-down gland phenotypes, which vary from necrotic tissue and irregular reservoir size to irregular color or separation of the secreted gland compounds. The knock-down of 13 genes caused specifically a strong reduction of para-benzoquinones, suggesting a specific function in the synthesis of these toxic compounds. Only 14 of the 69 confirmed gland genes are differentially overexpressed in stink gland tissue and thus could have been detected in a transcriptome-based analysis. Moreover, of the 29 previously transcriptomics-identified genes causing a gland phenotype, only one gene was recognized by this phenotypic screen despite the fact that 13 of them were covered by the screen. Conclusion Our results indicate the importance of combining diverse and independent methodologies to identify genes necessary for the function of a certain biological tissue, as the different approaches do not deliver redundant results but rather complement each other. The presented phenotypic screen together with a transcriptomics approach are now providing a set of close to hundred genes important for odoriferous defensive stink gland physiology in beetles.

scholarly journals Genome-Wide Identification of Essential and Auxiliary Gene Sets for Magnetosome Biosynthesis in Magnetospirillum gryphiswaldense

mSystems ◽  
2020 ◽  
Vol 5 (6) ◽  
Author(s):  
Karen T. Silva ◽  
Margarete Schüler ◽  
Frank Mickoleit ◽  
Theresa Zwiener ◽  
Frank D. Müller ◽  
...  
Keyword(s):  
Reverse Genetics ◽  
Intracellular Localization ◽  
Anaerobic Respiration ◽  
Magnetotactic Bacteria ◽  
Transposon Mutagenesis ◽  
Forward Genetics ◽  
Genetic Determinants ◽  
Content Type ◽  
Magnetospirillum Gryphiswaldense ◽  
Genome Wide

ABSTRACT Magnetotactic bacteria (MTB) stand out by their ability to manufacture membrane-enclosed magnetic organelles, so-called magnetosomes. Previously, it has been assumed that a genomic region of approximately 100 kbp, the magnetosome island (MAI), harbors all genetic determinants required for this intricate biosynthesis process. Recent evidence, however, argues for the involvement of additional auxiliary genes that have not been identified yet. In the present study, we set out to delineate the full gene complement required for magnetosome production in the alphaproteobacterium Magnetospirillum gryphiswaldense using a systematic genome-wide transposon mutagenesis approach. By an optimized procedure, a Tn5 insertion library of 80,000 clones was generated and screened, yielding close to 200 insertants with mild to severe impairment of magnetosome biosynthesis. Approximately 50% of all Tn5 insertion sites mapped within the MAI, mostly leading to a nonmagnetic phenotype. In contrast, in the majority of weakly magnetic Tn5 insertion mutants, genes outside the MAI were affected, which typically caused lower numbers of magnetite crystals with partly aberrant morphology, occasionally combined with deviant intracellular localization. While some of the Tn5-struck genes outside the MAI belong to pathways that have been linked to magnetosome formation before (e.g., aerobic and anaerobic respiration), the majority of affected genes are involved in so far unsuspected cellular processes, such as sulfate assimilation, oxidative protein folding, and cytochrome c maturation, or are altogether of unknown function. We also found that signal transduction and redox functions are enriched in the set of Tn5 hits outside the MAI, suggesting that such processes are particularly important in support of magnetosome biosynthesis. IMPORTANCE Magnetospirillum gryphiswaldense is one of the few tractable model magnetotactic bacteria (MTB) for studying magnetosome biomineralization. So far, knowledge on the genetic determinants of this complex process has been mainly gathered using reverse genetics and candidate approaches. In contrast, nontargeted forward genetics studies are lacking, since application of such techniques in MTB has been complicated for a number of technical reasons. Here, we report on the first comprehensive transposon mutagenesis study in MTB, aiming at systematic identification of auxiliary genes necessary to support magnetosome formation in addition to key genes harbored in the magnetosome island (MAI). Our work considerably extends the candidate set of novel subsidiary determinants and shows that the full gene complement underlying magnetosome biosynthesis is larger than assumed. In particular, we were able to define certain cellular pathways as specifically important for magnetosome formation that have not been implicated in this process so far.

scholarly journals Genetic analysis of Aedes aegypti captured at two international airports serving to the Greater Tokyo Area during 2012–2015

PLoS ONE ◽  
2020 ◽  
Vol 15 (4) ◽  
pp. e0232192
Author(s):  
Kentaro Itokawa ◽  
Jinping Hu ◽  
Nayu Sukehiro ◽  
Yoshio Tsuda ◽  
Osamu Komagata ◽  
...  
Keyword(s):  
Genetic Analysis ◽  
Aedes Aegypti

scholarly journals Genetic Identification of the Bacteriocins Produced by Enterococcus faecium IT62 and Evidence that Bacteriocin 32 Is Identical to Enterocin IT

2009 ◽  
Vol 53 (5) ◽  
pp. 1907-1911 ◽  
Author(s):  
Esther Izquierdo ◽  
Yimin Cai ◽  
Eric Marchioni ◽  
Saïd Ennahar
Keyword(s):  
Amino Acids ◽  
Genetic Analysis ◽  
Structural Gene ◽  
Enterococcus Faecium ◽  
Open Reading Frames ◽  
Genetic Identification ◽  
Leader Peptide ◽  
Sequencing Analysis ◽  
The One ◽  
Reading Frames

ABSTRACT Enterococcus faecium IT62, a strain isolated from ryegrass in Japan, produces three bacteriocins (enterocins L50A, L50B, and IT) that have been previously purified and the primary structures of which have been determined by amino acid sequencing (E. Izquierdo, A. Bednarczyk, C. Schaeffer, Y. Cai, E. Marchioni, A. Van Dorsselaer, and S. Ennahar, Antimicrob. Agents Chemother., 52:1917-1923, 2008). Genetic analysis showed that the bacteriocins of E. faecium IT62 are plasmid encoded, but with the structural genes specifying enterocin L50A and enterocin L50B being carried by a plasmid (pTAB1) that is separate from the one (pTIT1) carrying the structural gene of enterocin IT. Sequencing analysis of a 1,475-bp region from pTAB1 identified two consecutive open reading frames corresponding, with the exception of 2 bp, to the genes entL50A and entL50B, encoding EntL50A and EntL50B, respectively. Both bacteriocins are synthesized without N-terminal leader sequences. Genetic analysis of a sequenced 1,380-bp pTIT1 fragment showed that the genes entIT and entIM, encoding enterocin IT and its immunity protein, respectively, were both found in E. faecium VRE200 for bacteriocin 32. Enterocin IT, a 6,390-Da peptide made up of 54 amino acids, has been previously shown to be identical to the C-terminal part of bacteriocin 32, a 7,998-Da bacteriocin produced by E. faecium VRE200 whose structure was deduced from its structural gene (T. Inoue, H. Tomita, and Y. Ike, Antimicrob. Agents Chemother., 50:1202-1212, 2006). By combining the biochemical and genetic data on enterocin IT, it was concluded that bacteriocin 32 is in fact identical to enterocin IT, both being encoded by the same plasmid-borne gene, and that the N-terminal leader peptide for this bacteriocin is 35 amino acids long and not 19 amino acids long as previously reported.

scholarly journals Ultra-Low Volume Application of Spinosad (Natular 2EC) Larvicide as a Residual in a Tropical Environment Against Aedes and Anopheles Species

2018 ◽  
Vol 34 (1) ◽  
pp. 58-62 ◽  
Author(s):  
Seth C. Britch ◽  
Kenneth J. Linthicum ◽  
Robert L. Aldridge ◽  
Frances V. Golden ◽  
Arissara Pongsiri ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
Management Program ◽  
Dry Season ◽  
Tropical Environment ◽  
Integrated Vector Management ◽  
Anopheles Dirus ◽  
Important Species ◽  
Anopheles Species ◽  
Vector Management ◽  
Low Volume

ABSTRACT We investigated the efficacy of a liquid larvicide, Natular 2EC® (spinosad), applied with ultra-low volume sprayer as a residual application during the dry season in southeastern Thailand against 4 medically important species—Aedes aegypti, Ae. albopictus, Anopheles dirus, and An. minimus. We found that this larvicide could be applied as a residual to dry areas known to collect water and potentially still be effective after rains or irrigation, which could increase the flexibility and efficiency of an integrated vector management program targeting these species. This investigation also demonstrated, for the 1st time, efficacy of spinosad against An. minimus and An. dirus.

scholarly journals Development and characterization of microsatellite markers for two subspecies of Handroanthus chrysanthus

Rodriguésia ◽  
2021 ◽  
Vol 72 ◽  
Author(s):  
Augusta Yadira Cueva-Agila ◽  
Carlos Manchego ◽  
Cristian Bastidas ◽  
Manuel Curto
Keyword(s):  
Genetic Diversity ◽  
South America ◽  
Genetic Analysis ◽  
Microsatellite Markers ◽  
High Throughput Sequencing ◽  
Biodiversity Loss ◽  
Important Species ◽  
Polymorphic Microsatellites ◽  
Genetic Diversity And Structure

Abstract An understanding of the genetic diversity and structure of plant species is essential in order to comprehend the degree of biodiversity loss and to develop successful restoration programs. Handroanthus is an important genus that presents one of the most valuable timbers of South America. Handroanthus chrysanthus is an important species distributed in Central and South America. Microsatellite markers are not previously developed for this species. Ten microsatellites for Handroanthus chrysanthus developed using high-throughput sequencing are presented here. The usefulness of these microsatellite loci for the genetic analysis of subspecies H. chrysanthus subsp. chrysanthus (distributed in coastal dry forests) and subspecies H. chrysanthus subsp. meridionalis (distributed in premontane moist forests) is analyzed. At least eight polymorphic microsatellites are useful for each subspecies, seven of which can be used in both subspecies.

scholarly journals Transgenic expression of Nix converts genetic females into males and allows automated sex sorting in Aedes albopictus

2021 ◽  
Author(s):  
Célia Lutrat ◽  
Roenick Proveti Olmo ◽  
Thierry Baldet ◽  
Jérémy Bouyer ◽  
Eric Marois
Keyword(s):  
Aedes Aegypti ◽  
Aedes Albopictus ◽  
Mosquito Control ◽  
Flight Performance ◽  
Genetic Sexing ◽  
Transgenic Expression ◽  
Fluorescence Marker ◽  
Transgenic Lines ◽  
Genetic Sexing Strains ◽  
Primary Gene

Aedes albopictus is a major vector of arboviruses. Better understanding of its sex determination is crucial for developing mosquito control tools, especially genetic sexing strains. In Aedes aegypti, Nix is the primary gene responsible for masculinization and Nix-expressing genetic females develop into fertile, albeit flightless, males. In Ae. albopictus, Nix has also been implicated in masculinization but its role remains to be further characterized. In this work, we established Ae. albopictus transgenic lines ectopically expressing Nix. Several were composed exclusively of genetic females, with transgenic individuals being phenotypic and functional males due to the expression of the Nix transgene. Their reproductive fitness was marginally impaired, while their flight performance was similar to controls. Overall, our results show that Nix is sufficient for full masculinization in Ae. albopictus. Moreover, the transgene construct contains a fluorescence marker allowing efficient automated sex sorting. Consequently, such strains constitute valuable sexing strains for genetic control.

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