Development of sequence characterized amplified region (SCAR) markers linked to race-specific resistance to Striga gesnerioides in cowpea (Vigna unguiculata L.)

An amplified fragement length polymorphism (AFLP) fragment, E-ACT/M-CAA524, tightly linked to the Striga gesnerioides race 1 (SG1) resistance gene Rsg-2-1 in cowpea (Vigna unguiculata L.) was isolated by polyacrylamide gel electrophoresis, cloned, and its nucleotide sequence determined. Based on the resulting sequence information, a pair of sequence specific primers were designed and used to isolate identical and similar fragments from cowpea genomic DNA of different cowpea lines by polymerase chain reaction (PCR) amplification. The primers amplified a ~500 bp fragment (SCAR marker designated as 61R) that was present in the resistant parent TVU14676, absent in susceptible parent IT84S-2246, and segregated with the resistance phenotype in an F2 population, derived from a cross of these two genotypes. The same primers were used to isolate a fragment similar to 61R from another S. gesnerioides resistant line Kvx 61-1. The sequence of this fragment was used to design a new combination of primers that developed a second SCAR marker, designated as 61R-M2. Subsequent analysis of the three markers, E-ACT/M-CAA524, 61R and 61M2 showed that they are linked to each other by 0.6 centimorgans (cM). The utility of these SCARs in marker assisted selection programs for cowpea was discussed.Keywords: Striga gesnerioides, centimorgans (cM), race specific resistance, amplified fragment length polymorphism (AFLP), sequence characterized amplified region (SCAR), marker assisted selection (MAS

Flash Cracking Reactor for Waste Plastic Processing

Conversion of waste plastic to energy is a growing problem that is especially acute in space exploration applications. Moreover, utilization of heavy hydrocarbon resources (wastes, waxes, etc.) as fuels and chemicals will be a growing need in the future. Existing technologies require a trade-off between product selectivity and feedstock conversion. The objective of this work was to maintain high plastic-to-fuel conversion without sacrificing the liquid yield. The developed technology accomplishes this goal with a combined understanding of thermodynamics, reaction rates, and mass transport to achieve high feed conversion without sacrificing product selectivity. The innovation requires a reaction vessel, hydrocarbon feed, gas feed, and pressure and temperature control equipment. Depending on the feedstock and desired product distribution, catalyst can be added. The reactor is heated to the desired tempera ture, pressurized to the desired pressure, and subject to a sweep flow at the optimized superficial velocity. Software developed under this project can be used to determine optimal values for these parameters. Product is vaporized, transferred to a receiver, and cooled to a liquid - a form suitable for long-term storage as a fuel or chemical. An important NASA application is the use of solar energy to convert waste plastic into a form that can be utilized during periods of low solar energy flux. Unlike previous work in this field, this innovation uses thermodynamic, mass transport, and reaction parameters to tune product distribution of pyrolysis cracking. Previous work in this field has used some of these variables, but never all in conjunction for process optimization. This method is useful for municipal waste incinerator operators and gas-to-liquids companies

Combining Experiment and Theory to Elucidate the Role of Supercritical Water in Sulfide Decomposition

The cleavage of C–S linkages plays a key role in fuel processing and organic geochemistry. Water is known to affect these processes, and several hypotheses have been proposed, but the mechanism has been elusive. Here we use both experiment and theory to demonstrate that supercritical water reacts with intermediates formed during alkyl sulfide decomposition. During hexyl sulfide decomposition in supercritical water, pentane and CO + CO2 were detected in addition to the expected six carbon products. A multi-step reaction sequence for hexyl sulfide reacting with supercritical water is proposed which explains the surprising products, and quantum chemical calculations provide quantitative rates that support the proposed mechanism. The key sequence is cleavage of one C–S bond to form a thioaldehyde via radical reactions, followed by a pericyclic addition of water to the CS bond to form a geminal mercaptoalcohol. The mercaptoalcohol decomposes into an aldehyde and H2S either directly or via a water-catalyzed 6-membered ring transition state. The aldehyde quickly decomposes into CO plus pentane by radical reactions. The time is ripe for quantitative modelling of organosulfur reaction kinetics based on modern quantum chemistry

Engineering of an environmental isolate of bacillus megaterium for biochemical production under supercritical CO2

Continuous processing is a mainstay for chemical production but is far less common for biochemical processes. The increase in productivity and corresponding decrease in costs make continuous processing an intriguing option for bulk chemicals where price is a major consideration. Among the various challenges of continuous bioprocessing are the risks of contamination and the toxicity of the target products. Supercritical carbon dioxide (scCO2) may provide a means to address both of these issues. scCO2 is an attractive substitute for conventional organic solvents due to its unique transport and thermodynamic properties, its renewability and labile nature, and its high solubility for compounds such as alcohols, ketones and aldehydes. scCO2 is also known for its broad microbial lethality. The isolation and engineering of a microbe that is capable of growth and production in the presence of scCO2 thus represents an opportunity to create a production environment that is both resist to contamination and capable of sequestering toxic products through phase separation. Using a targeted bioprospecting approach by sampling fluid from a natural, deep-subsurface scCO2 well, a strain of Bacillus megaterium was isolated that is able to germinate and grow in the presence of scCO2. Transformation is possible using a protoplast-based method, which permitted the identification of promoters capable of inducible heterologous protein expression in both aerobic and anaerobic conditions. A xylose-inducible promoter was evaluated under scCO2 and found to have similar expression under both conditions. We engineered the B. megaterium strain to produce isobutanol from 2-ketoisovalerate by introducing a two-enzyme pathway (2- ketoisovalerate decarboxylase (KivD) and alcohol dehydrogenase (Adh)). Due to the strong partition of the aldehyde to the scCO2 phase, we tested five homologous Adh enzymes and found that YqhD from E. coli resulted in greater than 85% conversion when grown aerobically. Isobutanol production was also observed when our recombinant strain was cultured under scCO2. Finally, we have developed a process model for an integrated bioprocess and have found conditions that are comparable if not better than existing in situ extraction techniques such as gas stripping. Boock, J.T., A.J.E. Freedman, G.A. Tompsett, S.K. Muse, A.J. Allen, L.A. Jackson, B. Castro-Dominguez, M.T. Timko, K.L.J. Prather (co-corresponding author), J.R. Thompson. 2019. “Engineered microbial biofuel production and recovery under supercritical carbon dioxide.” Nat. Commun. 10:587. DOI: 10.1038/s41467- 019-08486-6

TOBFAC: the database of tobacco transcription factors

<p>Abstract</p> <p>Background</p> <p>Regulation of gene expression at the level of transcription is a major control point in many biological processes. Transcription factors (TFs) can activate and/or repress the transcriptional rate of target genes and vascular plant genomes devote approximately 7% of their coding capacity to TFs. Global analysis of TFs has only been performed for three complete higher plant genomes – Arabidopsis (<it>Arabidopsis thaliana</it>), poplar (<it>Populus trichocarpa</it>) and rice (<it>Oryza sativa</it>). Presently, no large-scale analysis of TFs has been made from a member of the <it>Solanaceae</it>, one of the most important families of vascular plants. To fill this void, we have analysed tobacco (<it>Nicotiana tabacum</it>) TFs using a dataset of 1,159,022 gene-space sequence reads (GSRs) obtained by methylation filtering of the tobacco genome. An analytical pipeline was developed to isolate TF sequences from the GSR data set. This involved multiple (typically 10–15) independent searches with different versions of the TF family-defining domain(s) (normally the DNA-binding domain) followed by assembly into contigs and verification. Our analysis revealed that tobacco contains a minimum of 2,513 TFs representing all of the 64 well-characterised plant TF families. The number of TFs in tobacco is higher than previously reported for Arabidopsis and rice.</p> <p>Results</p> <p>TOBFAC: the database of tobacco transcription factors, is an integrative database that provides a portal to sequence and phylogeny data for the identified TFs, together with a large quantity of other data concerning TFs in tobacco. The database contains an individual page dedicated to each of the 64 TF families. These contain background information, domain architecture via Pfam links, a list of all sequences and an assessment of the minimum number of TFs in this family in tobacco. Downloadable phylogenetic trees of the major families are provided along with detailed information on the bioinformatic pipeline that was used to find all family members. TOBFAC also contains EST data, a list of published tobacco TFs and a list of papers concerning tobacco TFs. The sequences and annotation data are stored in relational tables using a PostgrelSQL relational database management system. The data processing and analysis pipelines used the Perl programming language. The web interface was implemented in JavaScript and Perl CGI running on an Apache web server. The computationally intensive data processing and analysis pipelines were run on an Apple XServe cluster with more than 20 nodes.</p> <p>Conclusion</p> <p>TOBFAC is an expandable knowledgebase of tobacco TFs with data currently available for over 2,513 TFs from 64 gene families. TOBFAC integrates available sequence information, phylogenetic analysis, and EST data with published reports on tobacco TF function. The database provides a major resource for the study of gene expression in tobacco and the <it>Solanaceae </it>and helps to fill a current gap in studies of TF families across the plant kingdom. TOBFAC is publicly accessible at <url>http://compsysbio.achs.virginia.edu/tobfac/</url>.</p

Novel Catalysts and Processing Technologies for Production of Aerospace Fuels from Non-Petroleum Raw Materials

Transportation fuels production (including aerospace propellants) from non-traditional sources (gases, waste materials, and biomass) has been an active area of research and development for decades. Reducing terrestrial waste streams simultaneous with energy conversion, plentiful biomass, new low-cost methane sources, and/or extra-terrestrial resource harvesting and utilization present significant technological and business opportunities being realized by a new generation of visionary entrepreneurs. We examine several new approaches to catalyst fabrication and new processing technologies to enable utilization of these nontraditional raw materials. Two basic processing architectures are considered: a single-stage pyrolysis approach that seeks to basically re-cycle hydrocarbons with minimal net chemistry or a two-step paradigm that involves production of supply or synthesis gas (mainly carbon oxides and H2) followed by production of fuel(s) via Sabatier or methanation reactions and/or Fischer-Trpsch synthesis. Optimizing the fraction of product stream relevant to targeted aerospace (and other transportation) fuels via modeling, catalyst fabrication and novel reactor design are described. Energy utilization is a concern for production of fuels for either terrestrial or space operations; renewable sources based on solar energy and/or energy efficient processes may be mission enabling. Another important issue is minimizing impurities in the product stream(s), especially those potentially posing risks to personnel or operations through (catalyst) poisoning or (equipment) damage. Technologies being developed to remove (and/or recycle) heteroatom impurities are briefly discussed as well as the development of chemically robust catalysts whose activities are not diminished during operation. The potential impacts on future missions by such new approaches as well as balance of system issues are addressed

Sequencing and analysis of the gene-rich space of cowpea

<p>Abstract</p> <p>Background</p> <p>Cowpea, <it>Vigna unguiculata </it>(L.) Walp., is one of the most important food and forage legumes in the semi-arid tropics because of its drought tolerance and ability to grow on poor quality soils. Approximately 80% of cowpea production takes place in the dry savannahs of tropical West and Central Africa, mostly by poor subsistence farmers. Despite its economic and social importance in the developing world, cowpea remains to a large extent an underexploited crop. Among the major goals of cowpea breeding and improvement programs is the stacking of desirable agronomic traits, such as disease and pest resistance and response to abiotic stresses. Implementation of marker-assisted selection and breeding programs is severely limited by a paucity of trait-linked markers and a general lack of information on gene structure and organization. With a nuclear genome size estimated at ~620 Mb, the cowpea genome is an ideal target for reduced representation sequencing.</p> <p>Results</p> <p>We report here the sequencing and analysis of the gene-rich, hypomethylated portion of the cowpea genome selectively cloned by methylation filtration (MF) technology. Over 250,000 gene-space sequence reads (GSRs) with an average length of 610 bp were generated, yielding ~160 Mb of sequence information. The GSRs were assembled, annotated by BLAST homology searches of four public protein annotation databases and four plant proteomes (<it>A. thaliana</it>, <it>M. truncatula, O. sativa</it>, and <it>P. trichocarpa</it>), and analyzed using various domain and gene modeling tools. A total of 41,260 GSR assemblies and singletons were annotated, of which 19,786 have unique GenBank accession numbers. Within the GSR dataset, 29% of the sequences were annotated using the Arabidopsis Gene Ontology (GO) with the largest categories of assigned function being catalytic activity and metabolic processes, groups that include the majority of cellular enzymes and components of amino acid, carbohydrate and lipid metabolism. A total of 5,888 GSRs had homology to genes encoding transcription factors (TFs) and transcription associated factors (TAFs) representing about 5% of the total annotated sequences in the dataset. Sixty-two (62) of the 64 well-characterized plant transcription factor (TF) gene families are represented in the cowpea GSRs, and these families are of similar size and phylogenetic organization to those characterized in other plants. The cowpea GSRs also provides a rich source of genes involved in photoperiodic control, symbiosis, and defense-related responses. Comparisons to available databases revealed that about 74% of cowpea ESTs and 70% of all legume ESTs were represented in the GSR dataset. As approximately 12% of all GSRs contain an identifiable simple-sequence repeat, the dataset is a powerful resource for the design of microsatellite markers.</p> <p>Conclusion</p> <p>The availability of extensive publicly available genomic data for cowpea, a non-model legume with significant importance in the developing world, represents a significant step forward in legume research. Not only does the gene space sequence enable the detailed analysis of gene structure, gene family organization and phylogenetic relationships within cowpea, but it also facilitates the characterization of syntenic relationships with other cultivated and model legumes, and will contribute to determining patterns of chromosomal evolution in the Leguminosae. The micro and macrosyntenic relationships detected between cowpea and other cultivated and model legumes should simplify the identification of informative markers for marker-assisted trait selection and map-based gene isolation necessary for cowpea improvement.</p

Confirmation de QTL et validation de marqueurs SNPs associés à la résistance du niébé à Colletotrichum capsici, agent responsable de la maladie des taches brunes

Le niébé (Vigna unguiculata (L.) Walp.) est une légumineuse à graine très importante et constitue la principale source de protéines végétales pour l’alimentation des populations d’Afrique Subsaharienne. Sa production au Burkina Faso est entravée par la maladie des taches brunes provoquée par un champignon, Colletotrichum capsici (Syd.) Butler et Bisby. C’est dans la perspective d’accroître la productivité du niébé que nous avons entrepris de renforcer la lutte variétale contre cet agent pathogène. L’identification de marqueurs SNPs (Single Nucleotide Polymorphism) et QTL liés à la résistance à la maladie des taches brunes a été entrepris à partir d’une population biparentale F2 issus du croisement entre la variété sensible Tiligré et celle résistante KN-1. L’analyse QTL de la résistance du niébé à C. capsici à partir de la méthode ICIM add. a permis de confirmer et de valider respectivement un QTL majeur dénommé qBBDR2.1 et 9 marqueurs SNPs convertis, lesquels ont été cartographiés sur le chromosome Vu02 du niébé. Ce QTL dominant a présenté des effets additifs élevés liés aux allèles favorables de KN-1 et des valeurs de PVE de l’ordre de 51,50% et 55,33%, respectivement aux 21ème et 28ème JAI. English title: Confirmation of QTL mapping and validation of SNPs markers associated to cowpea resistance to Colletotrichum capsici, causal agent of brown blotch disease Cowpea (Vigna unguiculata (L.)Walp.) is one of the most important grain legume crops and constitutes the main source of plant protein for people food in sub-Saharan Africa. Cowpea production in Burkina Faso is constrained by brown blotch disease caused by a fungal,&nbsp; Colletotrichum capsici (Syd.) Butler and Bisby. In order to increase cowpea productivity we initiated a project to enhance host plant resistance to control the pathogen. The identification of SNP (Single Nucleotide Polymorphism) markers and QTL associated with brown blotch disease resistance was undertaken from a bi-parental F2 population resulting from a cross between the sensitive variety Tiligre and the resistant KN-1 to the disease. QTL analysis of cowpea resistance to C. capsici using the ICIM add method. Allowed to confirm and validate respectively a major QTL named qBBDR2.1 and 9 converted SNP markers, which were mapped on cowpea chromosome Vu02. This dominant QTL showed higher additive effects associated to alleles from KN-1 and PVE values of 51.50% and 55.33% respectively at 21 and 28 days after inoculatio