OPEN leaf: an open-source cloud-based phenotyping system for tracking dynamic changes at leaf-specific resolution in Arabidopsis

The first draft of the Arabidopsis genome was released more than 20 years ago and despite intensive molecular research, more than 30% of Arabidopsis genes remained uncharacterized or without an assigned function. This is in part due to gene redundancy within gene families or the essential nature of genes, where their deletion results in lethality (i.e., the dark genome). High-throughput plant phenotyping (HTPP) offers an automated and unbiased approach to characterize subtle or transient phenotypes resulting from gene redundancy or inducible gene silencing; however, commercial HTPP platforms remain unaffordable. Here we describe the design and implementation of OPEN leaf, an open-source HTPP system with cloud connectivity and remote bilateral communication to facilitate data collection, sharing and processing. OPEN leaf, coupled with the SMART imaging processing package was able to consistently document and quantify dynamic morphological changes over time at the whole rosette level and also at leaf-specific resolution when plants experienced changes in nutrient availability. The modular design of OPEN leaf allows for additional sensor integration. Notably, our data demonstrate that VIS sensors remain underutilized and can be used in high-throughput screens to identify characterize previously unidentified phenotypes in a leaf-specific manner.

Essential gene acquisition destabilizes plasmid inheritance

Extra-chromosomal genetic elements are important drivers of evolutionary transformations and ecological adaptations in prokaryotes with their evolutionary success often depending on their ‘utility’ to the host. Examples are plasmids encoding antibiotic resistance genes, which are known to proliferate in the presence of antibiotics. Plasmids carrying an essential host function are recognized as permanent residents in their host. Essential plasmids have been reported in several taxa where they often encode essential metabolic functions; nonetheless, their evolution remains poorly understood. Here we show that essential genes are rarely encoded on plasmids; evolving essential plasmids in Escherichia coli we further find that acquisition of an essential chromosomal gene by a plasmid can lead to plasmid extinction. A comparative genomics analysis of Escherichia isolates reveals few plasmid-encoded essential genes, yet these are often integrated into plasmid-related functions; an example is the GroEL/GroES chaperonin. Experimental evolution of a chaperonin-encoding plasmid shows that the acquisition of an essential gene reduces plasmid fitness regardless of the stability of plasmid inheritance. Our results suggest that essential plasmid emergence leads to a dose effect caused by gene redundancy. The detrimental effect of essential gene acquisition on plasmid inheritance constitutes a barrier for plasmid-mediated lateral gene transfer and supplies a mechanistic understanding for the rarity of essential genes in extra-chromosomal genetic elements.

An Improved CRISPR/Cas9 System for Genome Editing in Populus by Using Mannopine Synthase (MAS) Promoter

Gene editing technology in woody plants has great potential for understanding gene function, and altering traits affecting economically and ecologically important traits. Gene editing applications in woody species require a high genome editing efficiency due to the difficulty during transformation and complexities resulting from gene redundancy. In this study, we used poplar 84K (Populus alba × P. glandulosa), which is a model hybrid for studying wood formation and growth. We developed a new CRISPR/Cas9 system to edit multiple genes simultaneously. Using this system, we successfully knocked out multiple targets of the PHYTOENE DESATURASE 8 in poplar. We found the mutation rate of our CRISPR/Cas9 system is higher (67.5%) than existing reports in woody trees. We further improved the mutation rate up to 75% at editing sites through the usage of the mannopine synthase (MAS) promoter to drive Cas9. The MAS-CRISPR/Cas9 is an improved genome-editing tool for woody plants with a higher efficiency and a higher mutation rate than currently available technologies.

Impact of 16S rRNA gene redundancy and primer pair selection on the quantification and classification of oral microbiota in next-generation sequencing

Background: The identification, at least at the species level, is highly desirable in 16S rRNA sequencing-based studies of the oral microbiota. However, no study in the oral microbiology field has examined the impact of which primer pair is selected to detect redundant and matching amplicons. Consequently, our aims were to: 1) evaluate the number of 16S rRNA genes in the complete genomes of all the bacterial and archaeal species ever detected in the human oral cavity; and 2) assess how the use of different primer pairs would affect the detection and classification of redundant amplicons and matching amplicons (MA) from different taxa. Results: A total of 709 complete genomes (518 bacteria, 191 archaea) were downloaded from the NCBI database, and their complete 16S rRNA genes were extracted. 94.1% of oral bacteria and 52.59% of oral archaea had more than one 16S rRNA gene in their respective genomes. Next, 33 primer pairs identified in previous research and 6 commonly used in the literature were used against all the genomes to obtain amplicons. Between 46.67%-1.29% of the bacterial species and between 38.89%-4.65% of the archaeal species had MA, affecting relevant genera present in the oral environment such as Actinomyces, Fusobacterium, Lactobacillus, Methanosarcina, Staphylococcus, and Streptococcus. The best primer pairs were (the species coverage with no MA values, SC-NMA; region; primer pair position for Escherichia coli J01859.1): KP_F048-OP_R030 for bacteria (93.55%; 3-7; 342-1079), KP_F018-KP_R063 for archaea (89.63%; 3-9; undefined-1506), and OP_F114_OP_R121 for both bacteria and archaea (92.52%; 3-9; 340-1405). Conclusions : In addition to the 16S rRNA gene redundancy, the considerable presence of matching amplicons must be controlled to ensure the accurate interpretation of microbial diversity data. The SC-NMA is a more useful parameter than the conventional coverage percentage for selecting the best primer pairs. The performance of the primer pairs to detect no MA species increases as the average length of the amplicons increases; none of these being the most widely used primer pairs in the oral literature. The choice of primer pair affects significantly diversity estimates and taxonomic classification, conditioning the comparability of oral microbiome studies using different primer pairs.

Lineage-level Divergence of Copepod Glycerol Transporters and the Emergence of Isoform-specific Trafficking Regulation

Transmembrane conductance of glycerol is typically facilitated by aquaglyceroporins (Glps), which are commonly encoded by multiple genes in metazoan organisms. To date, however, little is known concerning the evolution of Glps in Crustacea or what forces might underly such gene redundancy. Here we show that Glp evolution in Crustacea is highly divergent, ranging from single copy genes in species of tadpole shrimps, isopods, amphipods and decapods to up to 10 copies in diplostracan water fleas although with monophyletic origins in each lineage. By contrast Glp evolution in Copepoda appears to be polyphyletic, with high rates of gene duplication occurring in a genera- and species-specifc manner. Based upon functional experiments on the Glps from a parasitic copepod (Lepeophtheirus salmonis), we show that such lineage-level gene duplication and splice variation is coupled with a high rate of neofunctionalization. For L. salmonis, splice variation of a given gene resulted in tissue- or sex-specific expression of the channels, with each variant evolving unique sites for PKC or PKA regulation of intracellular membrane trafficking. The data thus reveal that mutations favouring a high fidelity control of intracellular trafficking regulation can be a selection force for the evolution and retention of multiple Glps in copepods.

The Evolution of the Mammalian ABCA6-like Genes: Analysis of Phylogenetic, Expression, and Population Genetic Data Reveals Complex Evolutionary Histories

ABC membrane transporters are a large and complex superfamily of ATP-binding cassette transporters that are present in all domains of life. Both their essential function and complexity are reflected by their retention across large expanses of organismal diversity and by the extensive expansion of individual members and subfamilies during evolutionary history. This expansion has resulted in the diverse ABCA transporter family that has in turn evolved into multiple subfamilies. Here, we focus on the ABCA6-like subfamily of ABCA transporters with the goal of understanding their evolutionary history including potential functional changes in, or loss of, individual members. Our analysis finds that ABCA6-like genes, consisting of ABCA6, 8, 9, and 10, are absent from representatives of both monotremes and marsupials and thus the duplications that generated these families most likely occurred at the base of the Eutherian or placental mammals. We have found evidence of both positive and relaxed selection among the ABCA6-like genes, suggesting dynamic changes in function and the potential of gene redundancy. Analysis of the ABCA10 genes further suggests that this gene has undergone relaxed selection only within the human lineage. These findings are complemented by human population data, where we observe an excess of deactivating homozygous mutations. We describe the complex evolutionary history of this ABCA transporter subfamily and demonstrate through the combination of evolutionary and population genetic analysis that ABCA10 is undergoing pseudogenization within humans.

Systematic tools for reprogramming plant gene expression in a simple model, Marchantia polymorpha

ABSTRACTWe present the OpenPlant toolkit, a set of interlinked resources and techniques to develop Marchantia as testbed for bioengineering in plants. Marchantia is a liverwort, a simple plant with an open form of development that allows direct visualization of gene expression and dynamics of cellular growth in living tissues. We describe new techniques for simple and efficient axenic propagation and maintenance of Marchantia lines with no requirement for glasshouse facilities. Marchantia plants spontaneously produce clonal propagules within a few weeks of regeneration, and lines can be amplified million-fold in a single generation by induction of the sexual phase of growth, crossing and harvesting of progeny spores. The plant has a simple morphology and genome with reduced gene redundancy, and the dominant phase of its life cycle is haploid, making genetic analysis easier. We have built robust Loop assembly vector systems for nuclear and chloroplast transformation and genome editing. These have provided the basis for building and testing a modular library of standardized DNA elements with highly desirable properties. We have screened transcriptomic data to identify a range of candidate genes, extracted putative promoter sequences, and tested them in vivo to identify new constitutive promoter elements. The resources have been combined into a toolkit for plant bioengineering that is accessible for laboratories without access to traditional facilities for plant biology research. The toolkit is being made available under the terms of the OpenMTA and will facilitate the establishment of common standards and the use of this simple plant as testbed for synthetic biology.

Composition of Feature Relevancy Based Biomarker Gene Selection in Gene Expression Dataset

Cancer gene selection plays a prominent work in the area of Bioinformatics. Gene selection methods aim to retain relevant genes and remove redundant genes. This proposed technique deals on gene selection techniques based on information theory. By investigating the information theory based on composition of feature relevancy, we consider that a excellent gene technique method could boost novel classification of the cancer gene data while reducing gene redundancy. Therefore, a modified gene selection technique called Composition of Feature Relevancy (CFR) is carried out. To assess CFR, the experiments are carrying out on five real-world cancer gene expression data sets and three best classifiers (KNN, Support Vector Machine and Random forest). The modified gene selection technique gives best outcome when competing to other recent technique in terms of accuracy and sensitivity in classification.

Testosterone Degradative Pathway of Novosphingobium tardaugens

In this work, we have shown that Novosphingobium tardaugens NBRC 16725 (strain ARI-1), a bacterial strain that was isolated due to its capacity to mineralize the estrogenic endocrine compound 17β-estradiol, is also able to mineralize testosterone, the androgenic endocrine compound. Using in silico analysis, we predicted a new putative steroid degradation (SD) gene cluster in strain ARI-1, which resembles genes involved in testosterone degradation in Comamonas testosteroni and other testosterone degrading bacteria like Actinobacteria (like Rhodococcus and Mycobacteria genera) although with significant differences in gene organization. A whole transcriptomic analysis of N. tardaugens revealed that testosterone produces a limited induction of the genes of the SD cluster that show a high basal expression in its absence. The 3β/17β-hydroxysteroid dehydrogenase involved in the first metabolic step of testosterone degradation was identified by using genetic and biochemical approaches. The construction of knockout mutant strains in the genes of the SD cluster together with in silico analyses suggests the existence of gene redundancy in the genome of N. tardaugens. This work will expand the knowledge about the metabolic pathways and biotransformation capabilities of a Gram-negative bacterium that could become a new model system in the bacterial steroid degradation field.