scholarly journals Loss of MAR1 Function is a Marker for Co-Selection of CRISPR-Induced Mutations in Plants

2021 ◽  
Vol 3 ◽  
Author(s):  
Jannis Rinne ◽  
Claus-Peter Witte ◽  
Marco Herde
Keyword(s):  
Tissue Culture ◽  
Plant Species ◽  
Target Gene ◽  
Iron Homeostasis ◽  
Marker Gene ◽  
Single Copy ◽  
Kanamycin Resistance ◽  
Control Population ◽  
Induced Mutations ◽  
Selection Of

In this study, we describe the establishment of the knockout marker gene MAR1 for selection of CRISPR/Cas9-edited Arabidopsis seedlings and tomato explants in tissue culture. MAR1 encodes a transporter that is located in mitochondria and chloroplasts and is involved in iron homeostasis. It also opportunistically transports aminoglycoside antibiotics into these organelles and defects of the gene render plants insensitive to those compounds. Here, we show that mutations of MAR1 induced by the CRISPR system confer kanamycin-resistance to Arabidopsis plants and tomato tissues. MAR1 is single-copy in a variety of plant species and the corresponding proteins form a distinct phylogenetic clade allowing easy identification of MAR1 orthologs in different plants. We demonstrate that in multiplexing approaches, where Arabidopsis seedlings were selected via a CRISPR/Cas9-induced kanamycin resistance mediated by MAR1 mutation, a mutation in a second target gene was observed with higher frequency than in a control population only selected for the presence of the transgene. This so called co-selection has not been shown before to occur in plants. The technique can be employed to select for edited plants, which might be particularly useful if editing events are rare.

scholarly journals Gene markers for exon capture and phylogenomics in ray-finned fishes

2017 ◽  
Author(s):  
Jiamei Jiang ◽  
Hao Yuan ◽  
Xin Zheng ◽  
Qian Wang ◽  
Ting Kuang ◽  
...  
Keyword(s):  
Target Gene ◽  
Single Copy ◽  
A Posteriori ◽  
Protein Coding ◽  
Gene Markers ◽  
Taxonomic Level ◽  
Exon Capture ◽  
Phylogenomic Study ◽  
Generation Sequencing ◽  
Selection Of

AbstractGene capture coupled with the next generation sequencing has become one of the favorable methods in subsampling genomes for phylogenomic studies. Many target gene markers have been developed in plants, sharks, frogs, reptiles and others, but few have been reported in the ray-finned fishes. Here, we identified a suite of “single-copy” protein coding sequence (CDS) markers through comparing eight fish genomes, and tested them empirically in 83 species (33 families and 11 orders) of ray-finned fishes. Sorting through the markers according to their completeness and phylogenetic decisiveness in taxa tested resulted in a selection of 4,434 markers, which were proven to be useful in reconstructing phylogenies of the ray-finned fishes at different taxonomic level. We also proposed a strategy of refining baits (probes) design a posteriori based on empirical data. The markers that we have developed may fill a gap in the tool kit of phylogenomic study in vertebrates.

scholarly journals Chromosomal Integration of tcbChlorocatechol Degradation Pathway Genes as a Means of Expanding the Growth Substrate Range of Bacteria To Include Haloaromatics

2000 ◽  
Vol 66 (8) ◽  
pp. 3255-3261 ◽  
Author(s):  
Michael Klemba ◽  
Barbara Jakobs ◽  
Rolf-Michael Wittich ◽  
Dietmar Pieper
Keyword(s):  
Carbon Source ◽  
Gene Cluster ◽  
Gene Dosage ◽  
Single Copy ◽  
Degradation Pathway ◽  
Kanamycin Resistance ◽  
Chromosomal Integration ◽  
The Relationship ◽  
Subsequent Selection ◽  
Selection Of

ABSTRACT The tcbR-tcbCDEF gene cluster, coding for the chlorocatechol ortho-cleavage pathway inPseudomonas sp. strain P51, has been cloned into a Tn5-based minitransposon. The minitransposon carrying thetcb gene cluster and a kanamycin resistance gene was transferred to Pseudomonas putida KT2442, and chromosomal integration was monitored by selection either for growth on 3-chlorobenzoate or for kanamycin resistance. Transconjugants able to utilize 3-chlorobenzoate as a sole carbon source were obtained, although at a >100-fold lower frequency than kanamycin-resistant transconjugants. The vast majority of kanamycin-resistant transconjugants were not capable of growth on 3-chlorobenzoate. Southern blot analysis revealed that many transconjugants selected directly on 3-chlorobenzoate contained multiple chromosomal copies of the tcb gene cluster, whereas those selected for kanamycin resistance possessed a single copy. Subsequent selection of kanamycin resistance-selected single-copy transconjugants for growth on 3-chlorobenzoate yielded colonies capable of utilizing this carbon source, but no amplification of the tcb gene cluster was apparent. Introduction of two copies of the tcb gene cluster without prior 3-chlorobenzoate selection resulted in transconjugants able to grow on this carbon source. Expression of thetcb chlorocatechol catabolic operon in P. putida thus represents a useful model system for analysis of the relationship among gene dosage, enzyme expression level, and growth on chloroaromatic substrates.

scholarly journals Estimating the quality of eukaryotic genomes recovered from metagenomic analysis with EukCC

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Paul Saary ◽  
Alex L. Mitchell ◽  
Robert D. Finn
Keyword(s):  
Marker Gene ◽  
Single Copy ◽  
High Quality ◽  
Dynamic Selection ◽  
Microbial Eukaryotes ◽  
Gene Sets ◽  
Current Availability ◽  
Eukaryotic Genomes ◽  
Selection Of

Abstract Microbial eukaryotes constitute a significant fraction of biodiversity and have recently gained more attention, but the recovery of high-quality metagenomic assembled eukaryotic genomes is limited by the current availability of tools. To help address this, we have developed EukCC, a tool for estimating the quality of eukaryotic genomes based on the automated dynamic selection of single copy marker gene sets. We demonstrate that our method outperforms current genome quality estimators, particularly for estimating contamination, and have applied EukCC to datasets derived from two different environments to enable the identification of novel eukaryote genomes, including one from the human skin.

scholarly journals Estimating the quality of eukaryotic genomes recovered from metagenomic analysis

2019 ◽  
Author(s):  
Paul Saary ◽  
Alex L. Mitchell ◽  
Robert D. Finn
Keyword(s):  
Large Fraction ◽  
Marker Gene ◽  
Single Copy ◽  
Dynamic Selection ◽  
Microbial Eukaryotes ◽  
Gene Sets ◽  
Eukaryotic Genomes ◽  
Eukaryote Genome ◽  
Selection Of

AbstractEukaryotes make up a large fraction of microbial biodiversity. However, the field of metagenomics has been heavily biased towards the study of just the prokaryotic fraction. This focus has driven the necessary methodological developments to enable the recovery of prokaryotic genomes from metagenomes, which has reliably yielded genomes from thousands of novel species. More recently, microbial eukaryotes have gained more attention, but there is yet to be a parallel explosion in the number of eukaryotic genomes recovered from metagenomic samples. One of the current deficiencies is the lack of a universally applicable and reliable tool for the estimation of eukaryote genome quality. To address this need, we have developed EukCC, a tool for estimating the quality of eukaryotic genomes based on the dynamic selection of single copy marker gene sets, with the aim of applying it to metagenomics datasets. We demonstrate that our method outperforms current genome quality estimators and have applied EukCC to datasets from two different biomes to enable the identification of novel genomes, including a eukaryote found on the human skin and a Bathycoccus species obtained from a marine sample.

A novel T-DNA vector design for selection of transgenic lines with simple transgene integration and stable transgene expression

2005 ◽  
Vol 32 (8) ◽  
pp. 671 ◽  
Author(s):  
Song Chen ◽  
Christopher A. Helliwell ◽  
Li-Min Wu ◽  
Elizabeth S. Dennis ◽  
Narayana M. Upadhyaya ◽  
...  
Keyword(s):  
Transgene Expression ◽  
Marker Gene ◽  
Direct Repeat ◽  
Transgene Silencing ◽  
Selective Marker ◽  
Hairpin Rna ◽  
Transgenic Lines ◽  
Flanking Sequences ◽  
Dna Vector ◽  
Selection Of

Plants transformed with Agrobacterium frequently contain T-DNA concatamers with direct-repeat (d / r) or inverted-repeat (i / r) transgene integrations, and these repetitive T-DNA insertions are often associated with transgene silencing. To facilitate the selection of transgenic lines with simple T-DNA insertions, we constructed a binary vector (pSIV) based on the principle of hairpin RNA (hpRNA)-induced gene silencing. The vector is designed so that any transformed cells that contain more than one insertion per locus should generate hpRNA against the selective marker gene, leading to its silencing. These cells should, therefore, be sensitive to the selective agent and less likely to regenerate. Results from Arabidopsis and tobacco transformation showed that pSIV gave considerably fewer transgenic lines with repetitive insertions than did a conventional T-DNA vector (pCON). Furthermore, the transgene was more stably expressed in the pSIV plants than in the pCON plants. Rescue of plant DNA flanking sequences from pSIV plants was significantly more frequent than from pCON plants, suggesting that pSIV is potentially useful for T-DNA tagging. Our results revealed a perfect correlation between the presence of tail-to-tail inverted repeats and transgene silencing, supporting the view that read-through hpRNA transcript derived from i / r T-DNA insertions is a primary inducer of transgene silencing in plants.

scholarly journals Prioritization and Selection of High Biomass Energy Producing Plant Species at Boset District, Central Ethiopia: An Ethnobotanical Approach 

2021 ◽  
Author(s):  
Tinsae Bahru Yifru ◽  
Berhane Kidane ◽  
Amsalu Tolessa
Keyword(s):  
Plant Species ◽  
Sustainable Use ◽  
Biomass Energy ◽  
Calotropis Procera ◽  
Sampled Data ◽  
High Biomass ◽  
Structured Interviews ◽  
Acacia Senegal ◽  
Selection Of Species ◽  
Selection Of

Abstract Background: In Ethiopia, about 92.3% of all the biomass energy is consumed by domestic households and the demand is growing from 10-14%. However, there are little/no practical experiences or documented indigenous knowledge on how traditional people identify and select high biomass producing plant species with short rotation periods at Boset District. Therefore, the present study was aimed at: (1) selecting and documenting high biomass energy producing plant species at Boset District; (2) identifying major predictor variables that influence the prioritization and selection of species; and (3) develop a Generalized Linear Model (GLM) to predict the selection of species. Methods: A total of 96 informants comprising 59 men and 37 women between the ages of 18 and 81 were sampled. Data were collected using structured interviews, guided field walk, discussions and field observations. Results: Collected data indicated that 88.5% of the informants involved in firewood collection, while 90% practiced charcoal making. A total of 1533.60 Birr per household on average was earned annually from this activity. A total of 25 firewood and/or charcoal plant species were identified and documented at Boset District. Of these, Acacia senegal, Acacia tortilis and Acacia robusta were the three best prioritized and selected indigenous high biomass producing species. Prosopis juliflora, Parthenium hysterophorus, Azadirachta indica, Calotropis procera, Cryptostegia grandiflora, Lantana camara and Senna occidentalis further grouped under introduced fuelwood species. Prediction of GLM assured sampled Kebeles and source of income generated from fuelwood species positively and significantly (p<0.001) related to selection of species. Higher efficiency to provide energy and heat; little or no smoke or soot; easier to cut and split the wood and easier availability were some of the main selection criteria. Conclusions: This study provides valuable information in selection and documenting of high biomass producing plant species for proper management and sustainable use at Boset District. The three most selected species (A. senegal, A. tortilis and A. robusta) should be further evaluated at laboratory to determine their energy values.

scholarly journals Inactivating Gene Expression with Antisense Modified Oligonucleotides

Acta Naturae ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 101-105
Author(s):  
Sidney Altman ◽  
Carlos Angele-Martinez
Keyword(s):  
Gene Expression ◽  
Tissue Culture ◽  
Plasmodium Falciparum ◽  
Human Tissue ◽  
Target Gene ◽  
Modified Oligonucleotides ◽  
Modified Nucleotides ◽  
Culture Cells ◽  
Tissue Culture Cells

Modified nucleotides, including phosphoramidates and mesyl nucleotides, are very effective in inactivating gene expression in bacteria. Gyr A is the target gene in several organisms, including Plasmodium falciparum. Antisense reactions with bacteria infecting citrus plants are promising but incomplete. Human tissue culture cells assayed with a different target are also susceptible to the presence of mesyl oligonucleotides.

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