Dually biofortified cisgenic tomatoes with increased flavonoids and branched-chain amino acids content.

Higher dietary intakes of flavonoids may have a beneficial role in cardiovascular disease prevention. Additionally, supplementation of branched-chain amino acids (BCAAs) in vegan diets can reduce risks associated to their deficiency, particularly in older adults, which can cause loss of skeletal muscle strength and mass. Most plant-derived foods contain only small amounts of BCAAs, and those plants with high levels of flavonoids are not eaten broadly. Here we describe the generation of metabolically engineered cisgenic tomatoes enriched in both flavonoids and BCAAs. In this approach, coding and regulatory DNA elements, all derived from the tomato genome, were combined to obtain a herbicide-resistant version of an acetolactate synthase (mSlALS) gene expressed broadly and a MYB12-like transcription factor (SlMYB12) expressed in a fruit-specific manner. The mSlALS played a dual role, as a selectable marker as well as being key enzyme in BCAA enrichment. The resulting cisgenic tomatoes were highly enriched in Leucine (21-fold compared to wild-type levels), Valine (ninefold) and Isoleucine (threefold) and concomitantly biofortified in several antioxidant flavonoids including kaempferol (64-fold) and quercetin (45-fold). Comprehensive metabolomic and transcriptomic analysis of the biofortified cisgenic tomatoes revealed marked differences to wild type and could serve to evaluate the safety of these biofortified fruits for human consumption.

Early Embryo Exposure to Assisted Reproductive Manipulation Induced Subtle Changes in Liver Epigenetics with No Apparent Negative Health Consequences in Rabbit.

Embryo manipulation is a requisite step in assisted reproductive technology (ART). Therefore, it is of great necessity to appraise the safety of ART and investigate the long-term effect, including lipid metabolism, on ART-conceived offspring. Augmenting our ART rabbit model to investigate lipid metabolic outcomes in offspring longitudinally, we detected variations in hepatic DNA methylation ART offspring in the F3 generation for embryonic exposure (multiple ovulation, vitrification and embryo transfer). Through adult liver metabolomics and proteomics, we identified changes mainly related to lipid metabolism (e.g., polyunsaturated fatty acids, steroids, steroid hormone). We also found that DNA methylation analysis was linked to changes in lipid metabolism and apoptosis genes. Nevertheless, these differences did not apparently alter the general health status. Thus, our findings suggest that ART is likely to be a player in embryo epigenetic events related to hepatic homeostasis alteration in adulthood.

Transcriptomic analysis of a near-isogenic line of melon with high fruit flesh firmness during ripening.

BACKGROUND: A near-isogenic line (NIL) of melon (SC10-2) with introgression in linkage group X was studied from harvest (at firm-ripe stage of maturity) until day 18 of postharvest storage at 20.5 °C together with its parental control ('Piel de Sapo', PS). RESULTS: SC10-2 showed higher flesh firmness and whole fruit hardness but lower juiciness than its parental. SC10-2 showed a decrease in respiration rate accompanied by a decrease in ethylene production during ripening, both of which fell to a greater extent than in PS. The introgression affected 11 volatile organic compounds (VOCs), the levels of which during ripening were generally higher in SC10-2 than in PS. Transcriptomic analysis from RNA-Seq revealed differentially expressed genes (DEGs) associated with the effects studied. For example, 909 DEGs were exclusive to the introgression, and only 23 DEGs were exclusive to postharvest ripening time. Major functions of the DEGs associated with introgression or ripening time were identified by cluster analysis. About 37 genes directly and/or indirectly affected the delay in ripening of SC10-2 compared with PS in general and, more particularly, the physiological and quality traits measured and, probably, the differential non-climacteric response. Of the former genes, we studied in more detail at least five that mapped in the introgression in linkage group (LG) X, and 32 outside it. CONCLUSION: There is an apparent control of textural changes, VOCs and fruit ripening by an expression quantitative trait locus located in LG X together with a direct control on them due to genes presented in the introgression (CmTrpD, CmNADH1, CmTCP15, CmGDSL esterase/lipase, and CmHK4-like) and CmNAC18. © 2020 Society of Chemical Industry.

Cold seawater induces early sexual developmental stages in the BPG axis of European eel males.

BACKGROUND: The impossibility of closing the life cycle of the European eel (Anguilla anguilla) in captivity troubles the future of this critically endangered species. In addition, the European eel is a highly valued and demanded resource, thus the successful closing of its life cycle would have a substantial economic and ecological impact. With the aim of obtaining the highest gamete quality, the study of the effects of environmental factors, such as temperature, on reproductive performance may prove valuable. This is especially true for the exposure to cold water, which has been reported to improve sexual development in multiple other Actinopterygii species. RESULTS: European eel males treated with cold seawater (10 °C, T10) for 2 weeks showed an increase in the proliferation and differentiation of spermatogonial cells until the differentiated spermatogonial type A cell stage, and elevated testosterone and 11-ketotestosterone plasma levels. Transcriptomes from the tissues of the brain-pituitary-gonad (BPG) axis of T10 samples revealed a differential gene expression profile compared to the other experimental groups, with clustering in a principal component analysis and in heat maps of all differentially expressed genes. Furthermore, a functional analysis of differentially expressed genes revealed enriched gene ontology terms involved in the regulation of circadian rhythm, histone modification, meiotic nuclear division, and others. CONCLUSIONS: Cold seawater treatment had a clear effect on the activity of the BPG-axis of European eel males. In particular, our cold seawater treatment induces the synchronization and increased proliferation and differentiation of specific spermatogonial cells. In the transcriptomic results, genes related to thermoception were observed. This thermoception may have caused the observed effects through epigenetic mechanisms, since all analysed tissues further revealed differentially expressed genes involved in histone modification. The presented results support our hypothesis that a low temperature seawater treatment induces an early sexual developmental stage in European eels. This hypothesis is logical given that the average temperature experienced by eels in the early stages of their oceanic reproductive migration is highly similar to that of this cold seawater treatment. Further studies are needed to test whether a cold seawater treatment can improve the response of European eels to artificial hormonal treatment, as the results suggest.

Assessment of Cas12a-mediated gene editing efficiency in plants.

The CRISPR/Cas12a editing system opens new possibilities for plant genome engineering. To obtain a comparative assessment of RNA-guided endonuclease (RGEN) types in plants, we adapted the CRISPR/Cas12a system to the GoldenBraid (GB) modular cloning platform and compared the efficiency of Acidaminococcus (As) and Lachnospiraceae (Lb) Cas12a variants with the previously described GB-assembled Streptococcus pyogenes Cas9 (SpCas9) constructs in eight Nicotiana benthamiana loci using transient expression. All three nucleases showed drastic target-dependent differences in efficiency, with LbCas12 producing higher mutagenesis rates in five of the eight loci assayed, as estimated with the T7E1 endonuclease assay. Attempts to engineer crRNA direct repeat (DR) had little effect improving on-target efficiency for AsCas12a and resulted deleterious in the case of LbCas12a. To complete the assessment of Cas12a activity, we carried out genome editing experiments in three different model plants, namely N. benthamiana, Solanum lycopersicum and Arabidopsis thaliana. For the latter, we also resequenced Cas12a-free segregating T2 lines to assess possible off-target effects. Our results showed that the mutagenesis footprint of Cas12a is enriched in deletions of -10 to -2 nucleotides and included in some instances complex rearrangements in the surroundings of the target sites. We found no evidence of off-target mutations neither in related sequences nor somewhere else in the genome. Collectively, this study shows that LbCas12a is a viable alternative to SpCas9 for plant genome engineering.

Comparative analysis of the Squamosa Promoter Binding-Like (SPL) gene family in Nicotiana benthamiana and Nicotiana tabacum.

SQUAMOSA PROMOTER BINDING-LIKE (SPL) proteins constitute a large family of transcription factors known to play key roles in growth and developmental processes, including juvenile-to-adult and vegetative-to-reproductive phase transitions. This makes SPLs interesting targets for precision breeding in plants of the Nicotiana genus used as e.g. recombinant biofactories. We report the identification of 49 SPL genes in Nicotiana tabacum cv. K326 and 43 SPL genes in Nicotiana benthamiana LAB strain, which were classified into eight phylogenetic groups according to the SPL classification in Arabidopsis. Exon-intron gene structure and DNA-binding domains were highly conserved between homeologues and orthologues. Thirty of the NbSPL genes and 33 of the NtSPL genes were found to be possible targets of microRNA 156. The expression of SPL genes in leaves was analysed by RNA-seq at three different stages, revealing that genes not under miR156 control were in general constitutively expressed at high levels, whereas miR156-regulated genes showed lower expression, often developmentally regulated. We selected the N. benthamiana SPL13_1a gene as target for a CRISPR/Cas9 knock-out experiment. We show here that a full knock-out in this single gene leads to a significant delay in flowering time, a trait that could be exploited to increase biomass for recombinant protein production.

A multi-omic Nicotiana benthamiana resource for fundamental research and biotechnology.

Nicotiana benthamiana is an invaluable model plant and biotechnology platform with a ~3 Gb allotetraploid genome. To further improve its usefulness and versatility, we have produced high-quality chromosome-level genome assemblies, coupled with transcriptome, epigenome, microRNA and transposable element datasets, for the ubiquitously used LAB strain and a related wild accession, QLD. In addition, single nucleotide polymorphism maps have been produced for a further two laboratory strains and four wild accessions. Despite the loss of five chromosomes from the ancestral tetraploid, expansion of intergenic regions, widespread segmental allopolyploidy, advanced diploidization and evidence of recent bursts of Copia pseudovirus (Copia) mobility not seen in other Nicotiana genomes, the two subgenomes of N. benthamiana show large regions of synteny across the Solanaceae. LAB and QLD have many genetic, metabolic and phenotypic differences, including disparate RNA interference responses, but are highly interfertile and amenable to genome editing and both transient and stable transformation. The LAB/QLD combination has the potential to be as useful as the Columbia-0/Landsberg errecta partnership, utilized from the early pioneering days of Arabidopsis genomics to today.

PYL1- and PYL8-like ABA Receptors of Nicotiana benthamiana Play a Key Role in ABA Response in Seed and Vegetative Tissue.

To face the challenges of climate change and sustainable food production, it is essential to develop crop genome editing techniques to pinpoint key genes involved in abiotic stress signaling. The identification of those prevailing abscisic acid (ABA) receptors that mediate plant-environment interactions is quite challenging in polyploid plants because of the high number of genes in the PYR/PYL/RCAR ABA receptor family. Nicotiana benthamiana is a biotechnological crop amenable to genome editing, and given the importance of ABA signaling in coping with drought stress, we initiated the analysis of its 23-member family of ABA receptors through multiplex CRISPR/Cas9-mediated editing. We generated several high-order mutants impaired in NbPYL1-like and NbPYL8-like receptors, which showed certain insensitivity to ABA for inhibition of seedling establishment, growth, and development of shoot and lateral roots as well as reduced sensitivity to the PYL1-agonist cyanabactin (CB). However, in these high-order mutants, regulation of transpiration was not affected and was responsive to ABA treatment. This reveals a robust and redundant control of transpiration in this allotetraploid plant that probably reflects its origin from the extreme habitat of central Australia.

In-Depth Characterization of greenflesh Tomato Mutants Obtained by CRISPR/Cas9 Editing: A Case Study With Implications for Breeding and Regulation.

Gene editing has already proved itself as an invaluable tool for the generation of mutants for crop breeding, yet its ultimate impact on agriculture will depend on how crops generated by gene editing technologies are regulated, and on our ability to characterize the impact of mutations on plant phenotype. A starting operational strategy for evaluating gene editing-based approaches to plant breeding might consist of assessing the effect of the induced mutations in a crop- and locus-specific manner: this involves the analysis of editing efficiency in different cultivars of a crop, the assessment of potential off-target mutations, and a phenotypic evaluation of edited lines carrying different mutated alleles. Here, we targeted the GREENFLESH (GF) locus in two tomato cultivars ('MoneyMaker' and 'San Marzano') and evaluated the efficiency, specificity and mutation patterns associated with CRISPR/Cas9 activity for this gene. The GF locus encodes a Mg-dechelatase responsible for initiating chlorophyll degradation; in gf mutants, ripe fruits accumulate both carotenoids and chlorophylls. Phenotypic evaluations were conducted on two transgene-free T2 'MoneyMaker' gf lines with different mutant alleles (a small insertion of 1 nucleotide and a larger deletion of 123 bp). Both lines, in addition to reduced chlorophyll degradation, showed a notable increase in carotenoid and tocopherol levels during fruit ripening. Infection of gf leaves and fruits with Botrytis cinerea resulted in a significant reduction of infected area and pathogen proliferation compared to the wild type (WT). Our data indicates that the CRISPR/Cas9-mediated mutation of the GF locus in tomato is efficient, specific and reproducible and that the resulting phenotype is robust and consistent with previously characterized greenflesh mutants obtained with different breeding techniques, while also shedding light on novel traits such as vitamin E overaccumulation and pathogen resistance. This makes GF an appealing target for breeding tomato cultivars with improved features for cultivation, as well as consumer appreciation and health.

K-seq, an affordable, reliable, and open Klenow NGS-based genotyping technology.

BACKGROUND: K-seq, a new genotyping methodology based on the amplification of genomic regions using two steps of Klenow amplification with short oligonucleotides, followed by standard PCR and Illumina sequencing, is presented. The protocol was accompanied by software developed to aid with primer set design. RESULTS: As the first examples, K-seq in species as diverse as tomato, dog and wheat was developed. K-seq provided genetic distances similar to those based on WGS in dogs. Experiments comparing K-seq and GBS in tomato showed similar genetic results, although K-seq had the advantage of finding more SNPs for the same number of Illumina reads. The technology reproducibility was tested with two independent runs of the tomato samples, and the correlation coefficient of the SNP coverages between samples was 0.8 and the genotype match was above 94%. K-seq also proved to be useful in polyploid species. The wheat samples generated specific markers for all subgenomes, and the SNPs generated from the diploid ancestors were located in the expected subgenome with accuracies greater than 80%. CONCLUSION: K-seq is an open, patent-unencumbered, easy-to-set-up, cost-effective and reliable technology ready to be used by any molecular biology laboratory without special equipment in many genetic studies.

The GB4.0 Platform, an All-In-One Tool for CRISPR/Cas-Based Multiplex Genome Engineering in Plants.

CRISPR/Cas ability to target several loci simultaneously (multiplexing) is a game-changer in plant breeding. Multiplexing not only accelerates trait pyramiding but also can unveil traits hidden by functional redundancy. Furthermore, multiplexing enhances dCas-based programmable gene expression and enables cascade-like gene regulation. However, the design and assembly of multiplex constructs comprising tandemly arrayed guide RNAs (gRNAs) requires scarless cloning and is still troublesome due to the presence of repetitive sequences, thus hampering a more widespread use. Here we present a comprehensive extension of the software-assisted cloning platform GoldenBraid (GB), in which, on top of its multigene cloning software, we integrate new tools for the Type IIS-based easy and rapid assembly of up to six tandemly-arrayed gRNAs with both Cas9 and Cas12a, using the gRNA-tRNA-spaced and the crRNA unspaced approaches, respectively. As stress tests for the new tools, we assembled and used for Agrobacterium-mediated stable transformation a 17 Cas9-gRNAs construct targeting a subset of the Squamosa-Promoter Binding Protein-Like (SPL) gene family in Nicotiana tabacum. The 14 selected genes are targets of miR156, thus potentially playing an important role in juvenile-to-adult and vegetative-to-reproductive phase transitions. With the 17 gRNAs construct we generated a collection of Cas9-free SPL edited T1 plants harboring up to 9 biallelic mutations and showing leaf juvenility and more branching. The functionality of GB-assembled dCas9 and dCas12a-based CRISPR/Cas activators and repressors using single and multiplexing gRNAs was validated using a Luciferase reporter with the Solanum lycopersicum Mtb promoter or the Agrobacterium tumefaciens nopaline synthase promoter in transient expression in Nicotiana benthamiana. With the incorporation of the new web-based tools and the accompanying collection of DNA parts, the GB4.0 genome edition turns an all-in-one open platform for plant genome engineering.

Long-term and transgenerational phenotypic, transcriptional and metabolic effects in rabbit males born following vitrified embryo transfer.

The advent of assisted reproductive technologies (ART) in mammals involved an extraordinary change in the environment where the beginning of a new organism takes place. Under in vitro conditions, in which ART is currently being performed, it likely fails to mimic optimal in vivo conditions. This suboptimal environment could mediate in the natural developmental trajectory of the embryo, inducing lasting effects until later life stages that may be inherited by subsequent generations (transgenerational effects). Therefore, we evaluated the potential transgenerational effects of embryo exposure to the cryopreservation-transfer procedure in a rabbit model on the offspring phenotype, molecular physiology of the liver (transcriptome and metabolome) and reproductive performance during three generations (F1, F2 and F3). The results showed that, compared to naturally-conceived animals (NC group), progeny generated after embryo exposure to the cryopreservation-transfer procedure (VT group) exhibited lower body growth, which incurred lower adult body weight in the F1 (direct effects), F2 (intergenerational effects) and F3 (transgenerational effects) generations. Furthermore, VT animals showed intergenerational effects on heart weight and transgenerational effects on liver weight. The RNA-seq data of liver tissue revealed 642 differentially expressed transcripts (DETs) in VT animals from the F1 generation. Of those, 133 were inherited from the F2 and 120 from the F3 generation. Accordingly, 151, 190 and 159 differentially accumulated metabolites (DAMs) were detected from the F1, F2 and F3, respectively. Moreover, targeted metabolomics analysis demonstrated that transgenerational effects were mostly presented in the non-polar fraction. Functional analysis of molecular data suggests weakened zinc and fatty acid metabolism across the generations, associated with alterations in a complex molecular network affecting global hepatic metabolism that could be associated with the phenotype of VT animals. However, these VT animals showed proper reproductive performance, which verified a functional health status. In conclusion, our results establish the long-term transgenerational effects following a vitrified embryo transfer procedure. We showed that the VT phenotype could be the result of the manifestation of embryonic developmental plasticity in response to the stressful conditions during ART procedures.

Exploiting the diversity of tomato: the development of a phenotypically and genetically detailed germplasm collection.

A collection of 163 accessions, including Solanum pimpinellifolium, Solanum lycopersicum var. cerasiforme and Solanum lycopersicum var. lycopersicum, was selected to represent the genetic and morphological variability of tomato at its centers of origin and domestication: Andean regions of Peru and Ecuador and Mesoamerica. The collection is enriched with S. lycopersicum var. cerasiforme from the Amazonian region that has not been analyzed previously nor used extensively. The collection has been morphologically characterized showing diversity for fruit, flower and vegetative traits. Their genomes were sequenced in the Varitome project and are publicly available (solgenomics.net/projects/varitome). The identified SNPs have been annotated with respect to their impact and a total number of 37,974 out of 19,364,146 SNPs have been described as high impact by the SnpEeff analysis. GWAS has shown associations for different traits, demonstrating the potential of this collection for this kind of analysis. We have not only identified known QTLs and genes, but also new regions associated with traits such as fruit color, number of flowers per inflorescence or inflorescence architecture. To speed up and facilitate the use of this information, F2 populations were constructed by crossing the whole collection with three different parents. This F2 collection is useful for testing SNPs identified by GWAs, selection sweeps or any other candidate gene. All data is available on Solanaceae Genomics Network and the accession and F2 seeds are freely available at COMAV and at TGRC genebanks. All these resources together make this collection a good candidate for genetic studies.

Multigene Engineering by GoldenBraid Cloning: From Plants to Filamentous Fungi and Beyond.

Many synthetic biologists have adopted methods based on Type IIS restriction enzymes and Golden Gate technology in their cloning procedures, as these enable the combinatorial assembly of modular elements in a very efficient way following standard rules. GoldenBraid (GB) is a Golden Gate-based modular cloning system that, in addition, facilitates the engineering of large multigene constructs and the exchange of DNA parts as result of its iterative cloning scheme. GB was initially developed specifically for plant synthetic biology, and it has been subsequently extended and adapted to other organisms such as Saccharomyces cerevisiae, filamentous fungi, and human cells by incorporating a number of host-specific features into its basic scheme. Here we describe the general GB cloning procedure and provide detailed protocols for its adaptation to filamentous fungi-a GB variant known as FungalBraid. The assembly of a cassette for gene disruption by homologous recombination, a fungal-specific extension of the GB utility, is also shown. Development of FungalBraid was relatively straightforward, as both plants and fungi can be engineered using the same binary plasmids via Agrobacterium-mediated transformation. We also describe the use of a set of web-based tools available at the GB website that assist users in all cloning procedures. The availability of plant and fungal versions of GB will facilitate genetic engineering in these industrially relevant organisms. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Software-assisted modular DNA assembly of a two gene expression-cassette with GB Basic Protocol 2: Agrobacterium tumefaciens-mediated transformation of filamentous fungi Basic Protocol 3: Software-assisted modular DNA assembly of a gene disruption-cassette using GB Basic Protocol 4: Obtaining disruption transformants.

De novo European eel transcriptome provides insights into the evolutionary history of duplicated genes in teleost lineages.

Paralogues pairs are more frequently observed in eels (Anguilla sp.) than in other teleosts. The paralogues often show low phylogenetic distances; however, they have been assigned to the third round of whole genome duplication (WGD), shared by all teleosts (3R), due to their conserved synteny. The apparent contradiction of low phylogenetic difference and 3R conserved synteny led us to study the duplicated gene complement of the freshwater eels. With this aim, we assembled de novo transcriptomes of two highly relevant freshwater eel species: The European (Anguilla anguilla) and the Japanese eel (Anguilla japonica). The duplicated gene complement was analysed in these transcriptomes, and in the genomes and transcriptomes of other Actinopterygii species. The study included an assessment of neutral genetic divergence (4dTv), synteny, and the phylogenetic origins and relationships of the duplicated gene complements. The analyses indicated a high accumulation of duplications (1217 paralogue pairs) among freshwater eel genes, which may have originated in a WGD event after the Elopomorpha lineage diverged from the remaining teleosts, and thus not at the 3R. However, very similar results were observed in the basal Osteoglossomorpha and Clupeocephala branches, indicating that the specific genomic regions of these paralogues may still have been under tetrasomic inheritance at the split of the teleost lineages. Therefore, two potential hypotheses may explain the results: i) The freshwater eel lineage experienced an additional WGD to 3R, and ii) Some duplicated genomic regions experienced lineage specific rediploidization after 3R in the ancestor to freshwater eels. The supporting/opposing evidence for both hypotheses is discussed.