Precise exogenous insertion and sequence replacements in poplar by simultaneous HDR overexpression and NHEJ suppression using CRISPR-Cas9

CRISPR-mediated genome editing has become a powerful tool for the genetic modification of biological traits. However, developing an efficient, site-specific, gene knock-in system based on homology-directed DNA repair (HDR) remains a significant challenge in plants, especially in woody species like poplar. Here, we show that simultaneous inhibition of non-homologous end joining (NHEJ) recombination cofactor XRCC4 and overexpression of HDR enhancer factors CtIP and MRE11 can improve HDR efficiency for gene knock-in. Using this approach, the BleoR gene was integrated onto the 3′ end of the MKK2 MAP kinase gene to generate a BleoR-MKK2 fusion protein. Based on fully edited nucleotides evaluated by TaqMan real-time PCR, the HDR-mediated knock-in efficiency was up to 48% when using XRCC4 silencing incorporated with a combination of CtIP and MRE11 overexpression compared with no HDR enhancement or NHEJ silencing. Furthermore, this combination of HDR enhancer overexpression and NHEJ repression also increased genome targeting efficiency and gave 7-fold fewer CRISPR-induced insertions and deletions (InDels), resulting in no functional effects on MKK2-based salt stress responses in poplar. Therefore, this approach may be useful not only in poplar and plants or crops but also in mammals for improving CRISPR-mediated gene knock-in efficiency

High-density genetic map using whole-genome resequencing for fine mapping and candidate gene discovery for disease resistance in peanut

Whole‐genome resequencing (WGRS) of mapping populations has facilitated development of high‐density genetic maps essential for fine mapping and candidate gene discovery for traits of interest in crop species. Leaf spots, including early leaf spot (ELS) and late leaf spot (LLS), and Tomato spotted wilt virus (TSWV) are devastating diseases in peanut causing significant yield loss. We generated WGRS data on a recombinant inbred line population, developed a SNP‐based high‐density genetic map, and conducted fine mapping, candidate gene discovery and marker validation for ELS, LLS and TSWV. The first sequence‐based high‐density map was constructed with 8869 SNPs assigned to 20 linkage groups, representing 20 chromosomes, for the ‘T’ population (Tifrunner × GT‐C20) with a map length of 3120 cM and an average distance of 1.45 cM. The quantitative trait locus (QTL) analysis using high‐density genetic map and multiple season phenotyping data identified 35 main‐effect QTLs with phenotypic variation explained (PVE) from 6.32% to 47.63%. Among major‐effect QTLs mapped, there were two QTLs for ELS on B05 with 47.42% PVE and B03 with 47.38% PVE, two QTLs for LLS on A05 with 47.63% and B03 with 34.03% PVE and one QTL for TSWV on B09 with 40.71% PVE. The epistasis and environment interaction analyses identified significant environmental effects on these traits. The identified QTL regions had disease resistance genes including R‐genes and transcription factors. KASP markers were developed for major QTLs and validated in the population and are ready for further deployment in genomics‐assisted breeding in peanut

Mitigating aflatoxin contamination in groundnut through a combination of genetic resistance and post-harvest management practices

Aflatoxin is considered a “hidden poison” due to its slow and adverse effect on various biological pathways in humans, particularly among children, in whom it leads to delayed development, stunted growth, liver damage, and liver cancer. Unfortunately, the unpredictable behavior of the fungus as well as climatic conditions pose serious challenges in precise phenotyping, genetic prediction and genetic improvement, leaving the complete onus of preventing aflatoxin contamination in crops on post-harvest management. Equipping popular crop varieties with genetic resistance to aflatoxin is key to effective lowering of infection in farmer’s fields. A combination of genetic resistance for in vitro seed colonization (IVSC), pre-harvest aflatoxin contamination (PAC) and aflatoxin production together with pre- and post-harvest management may provide a sustainable solution to aflatoxin contamination. In this context, modern “omics” approaches, including next-generation genomics technologies, can provide improved and decisive information and genetic solutions. Preventing contamination will not only drastically boost the consumption and trade of the crops and products across nations/regions, but more importantly, stave off deleterious health problems among consumers across the globe

Interconnecting Governments, Businesses and Citizens – A Comparison of Two Digital Infrastructures

Part 2: Services and InteroperabilityInternational audiencePublic and private organizations in various areas are setting up digital Information Infrastructures (IIs) for interconnecting government, businesses and citizens. IIs can create value by sharing and integrating data of multiple actors. This can be the basis for value added services and especially collaborations of public and private partners can make IIs thrive. Easier access to integrated services and products (jointly) offered by government and businesses may stimulate transparency and innovations. IIs are under development in many domains, including for open data and international trade. However, there are notable differences in the design, characteristics and implementation of the IIs. The objective of this paper is to compare two diverse IIs in order to obtain a better understanding of common and differing elements in the IIs and their impact. Among the differences are the roles of government, businesses and users, in driving, developing and exploitation of the IIs

A Pre-Landing Assessment of Regolith Properties at the InSight Landing Site

This article discusses relevant physical properties of the regolith at the Mars InSight landing site as understood prior to landing of the spacecraft. InSight will land in the northern lowland plains of Mars, close to the equator, where the regolith is estimated to be ≥3--5 m thick. These investigations of physical properties have relied on data collected from Mars orbital measurements, previously collected lander and rover data, results of studies of data and samples from Apollo lunar missions, laboratory measurements on regolith simulants, and theoretical studies. The investigations include changes in properties with depth and temperature. Mechanical properties investigated include density, grain-size distribution, cohesion, and angle of internal friction. Thermophysical properties include thermal inertia, surface emissivity and albedo, thermal conductivity and diffusivity, and specific heat. Regolith elastic properties not only include parameters that control seismic wave velocities in the immediate vicinity of the Insight lander but also coupling of the lander and other potential noise sources to the InSight broadband seismometer. The related properties include Poisson’s ratio, P- and S-wave velocities, Young’s modulus, and seismic attenuation. Finally, mass diffusivity was investigated to estimate gas movements in the regolith driven by atmospheric pressure changes. Physical properties presented here are all to some degree speculative. However, they form a basis for interpretation of the early data to be returned from the InSight mission.Additional co-authors: Nick Teanby and Sharon Keda

Carbohydrate, glutathione, and polyamine metabolism are central to Aspergillus flavus oxidative stress responses over time

Background The primary and secondary metabolites of fungi are critical for adaptation to environmental stresses, host pathogenicity, competition with other microbes, and reproductive fitness. Drought-derived reactive oxygen species (ROS) have been shown to stimulate aflatoxin production and regulate in Aspergillus flavus, and may function in signaling with host plants. Here, we have performed global, untargeted metabolomics to better understand the role of aflatoxin production in oxidative stress responses, and also explore isolate-specific oxidative stress responses over time. Results Two field isolates of A. flavus, AF13 and NRRL3357, possessing high and moderate aflatoxin production, respectively, were cultured in medium with and without supplementation with 15 mM H2O2, and mycelia were collected following 4 and 7 days in culture for global metabolomics. Overall, 389 compounds were described in the analysis which encompassed 9 biological super-pathways and 47 sub-pathways. These metabolites were examined for differential accumulation. Significant differences were observed in both isolates in response to oxidative stress and when comparing sampling time points. Conclusions The moderately high aflatoxin-producing isolate, NRRL3357, showed extensive stimulation of antioxidant mechanisms and pathways including polyamines metabolism, glutathione metabolism, TCA cycle, and lipid metabolism while the highly aflatoxigenic isolate, AF13, showed a less vigorous response to stress. Carbohydrate pathway levels also imply that carbohydrate repression and starvation may influence metabolite accumulation at the later timepoint. Higher conidial oxidative stress tolerance and antioxidant capacity in AF13 compared to NRRL3357, inferred from their metabolomic profiles and growth curves over time, may be connected to aflatoxin production capability and aflatoxin-related antioxidant accumulation. The coincidence of several of the detected metabolites in H2O2-stressed A. flavus and drought-stressed hosts also suggests their potential role in the interaction between these organisms and their use as markers/targets to enhance host resistance through biomarker selection or genetic engineering

Global transcriptome profiling identified transcription factors, biological process, and associated pathways for pre-harvest aflatoxin contamination in groundnut

Pre-harvest aflatoxin contamination (PAC) in groundnut is a serious quality concern globally, and drought stress before harvest further exacerbate its intensity, leading to the deterioration of produce quality. Understanding the host–pathogen interaction and identifying the candidate genes responsible for resistance to PAC will provide insights into the defense mechanism of the groundnut. In this context, about 971.63 million reads have been generated from 16 RNA samples under controlled and Aspergillus flavus infected conditions, from one susceptible and seven resistant genotypes. The RNA-seq analysis identified 45,336 genome-wide transcripts under control and infected conditions. This study identified 57 transcription factor (TF) families with major contributions from 6570 genes coding for bHLH (719), MYB-related (479), NAC (437), FAR1 family protein (320), and a few other families. In the host (groundnut), defense-related genes such as senescence-associated proteins, resveratrol synthase, seed linoleate, pathogenesis-related proteins, peroxidases, glutathione-S-transferases, chalcone synthase, ABA-responsive gene, and chitinases were found to be differentially expressed among resistant genotypes as compared to susceptible genotypes. This study also indicated the vital role of ABA-responsive ABR17, which co-regulates the genes of ABA responsive elements during drought stress, while providing resistance against A. flavus infection. It belongs to the PR-10 class and is also present in several plant–pathogen interactions

Extreme hydrochemical conditions in natural microcosms entombed within Antarctic ice

Cryoconite holes are near-vertical tubes that form in the surface of glaciers when solar-heated debris melts into the ice. Those that form in the McMurdo Dry Valleys of Antarctica are distinctive, in that they have ice lids and are closed to the atmosphere for periods of years to decades. Photoautotrophs and heterotrophs grow within this closed environment, perturbing the poorly buffered water chemistry, yet maintaining the potential for photosynthesis. Microbial excretion and decomposition of organic matter produces dissolved organic carbon (DOC): dissolved inorganic carbon ratios of ?1 : 2. Much of the dissolved nitrogen pool (80–100%) exists as dissolved organic nitrogen (DON). The DON:DOC ratio is ?1 : 11 (mol/mol), typical of organic particulate material at the Earth’s surface. The combination of photoautotrophy, heterotrophy and weak chemical buffering within these microcosms promotes values of pH, pCO2, O2 saturation and percentage total dissolved nitrogen as DON that reach 10·99, 10?7·6 atm, 160% and 100% respectively, which are a unique combination among the surface waters on Earth. These ice-sealed cryoconite holes could be important analogues of refugia on Snowball Earth and other icy planets