The Dark Side of the pollen: BSA-seq identified genomic regions linked to male sterility in globe artichoke.

Globe artichoke (Cynara cardunculus var. scolymus; 2n = 2x = 34) is a food crop consumed for its immature flower heads. Traditionally, globe artichoke varietal types are vegetatively propagated. However, seed propagation makes it possible to treat the crop as annual, increasing field uniformity and reducing farmers costs, as well as pathogens diffusion. Despite globe artichoke's significant agricultural value and the critical role of heterosis in the development of superior varieties, the production of hybrids remains challenging without a reliable system for large-scale industrial seed production. Male sterility (MS) presents a promising avenue for overcoming these challenges by simplifying the hybridization process and enabling cost-effective seed production. However, within the Cynara genus, genic male sterility has been linked to three recessive loci in globe artichoke, with no definitive genetic mechanism elucidated to date. A 250 offsprings F2 population, derived from a cross between a MS globe artichoke and a male fertile (MF) cultivated cardoon (C. cardunculus var. altilis) and fitting a monogenic segregation model (3:1), was analyzed through BSA-seq, aiming at the identification of genomic regions/genes affecting male sterility. Four QTL regions were identified on chromosomes 4, 12, and 14. By analyzing the sequence around the highest pick on chromosome 14, a cytochrome P450 (CYP703A2) was identified, carrying a deleterious substitution (R/Q) fixed in the male sterile parent. A single dCAPS marker was developed around this SNP, allowing the discrimination between MS and MF genotypes within the population, suitable for applications in plant breeding programs. A 3D model of the protein was generated by homology modeling, revealing that the mutated amino acid is part of a highly conserved motif crucial for protein folding.

Revitalizing agriculture: next-generation genotyping and -omics technologies enabling molecular prediction of resilient traits in the Solanaceae family.

This review highlights -omics research in Solanaceae family, with a particular focus on resilient traits. Extensive research has enriched our understanding of Solanaceae genomics and genetics, with historical varietal development mainly focusing on disease resistance and cultivar improvement but shifting the emphasis towards unveiling resilience mechanisms in genebank-preserved germplasm is nowadays crucial. Collecting such information, might help researchers and breeders developing new experimental design, providing an overview of the state of the art of the most advanced approaches for the identification of the genetic elements laying behind resilience. Building this starting point, we aim at providing a useful tool for tackling the global agricultural resilience goals in these crops.

Boosting grapevine breeding for climate-smart viticulture: from genetic resources to predictive genomics.

The multifaceted nature of climate change is increasing the urgency to select resilient grapevine varieties, or generate new, fitter cultivars, to withstand a multitude of new challenging conditions. The attainment of this goal is hindered by the limiting pace of traditional breeding approaches, which require decades to result in new selections. On the other hand, marker-assisted breeding has proved useful when it comes to traits governed by one or few genes with great effects on the phenotype, but its efficacy is still restricted for complex traits controlled by many loci. On these premises, innovative strategies are emerging which could help guide selection, taking advantage of the genetic diversity within the Vitis genus in its entirety. Multiple germplasm collections are also available as a source of genetic material for the introgression of alleles of interest via adapted and pioneering transformation protocols, which present themselves as promising tools for future applications on a notably recalcitrant species such as grapevine. Genome editing intersects both these strategies, not only by being an alternative to obtain focused changes in a relatively rapid way, but also by supporting a fine-tuning of new genotypes developed with other methods. A review on the state of the art concerning the available genetic resources and the possibilities of use of innovative techniques in aid of selection is presented here to support the production of climate-smart grapevine genotypes.

Structural and expression analysis of polyphenol oxidases potentially involved in globe artichoke (C. cardunculus var. scolymus L.) tissue browning.

Globe artichoke capitula are susceptible to browning due to oxidation of phenols caused by the activity of polyphenol oxidases (PPOs), this reduces their suitability for fresh or processed uses. A genome-wide analysis of the globe artichoke PPO gene family was performed. Bioinformatics analyses identified eleven PPOs and their genomic and amino acidic features were annotated. Cis-acting element analysis identified a gene regulatory and functional profile associated to plant growth and development as well as stress response. For some PPOs, phylogenetic analyses revealed a structural and functional conservation with different Asteraceae PPOs, while the allelic variants of the eleven PPOs investigated across four globe artichoke varietal types identified several SNP/Indel variants, some of which having impact on gene translation. By RTqPCR were assessed the expression patterns of PPOs in plant tissues and in vitro calli characterized by different morphologies. Heterogeneous PPO expression profiles were observed and three of them (PPO6, 7 and 11) showed a significant increase of transcripts in capitula tissues after cutting. Analogously, the same three PPOs were significantly up-regulated in calli showing a brown phenotype due to oxidation of phenols. Our results lay the foundations for a future application of gene editing aimed at disabling the three PPOs putatively involved in capitula browning.

Genomic Analysis Highlights Putative Defective Susceptibility Genes in Tomato Germplasm.

Tomato (Solanum lycopersicum L.) is one of the most widely grown vegetables in the world and is impacted by many diseases which cause yield reduction or even crop failure. Breeding for disease resistance is thus a key objective in tomato improvement. Since disease arises from a compatible interaction between a plant and a pathogen, a mutation which alters a plant susceptibility (S) gene facilitating compatibility may induce broad-spectrum and durable plant resistance. Here, we report on a genome-wide analysis of a set of 360 tomato genotypes, with the goal of identifying defective S-gene alleles as a potential source for the breeding of resistance. A set of 125 gene homologs of 10 S-genes (PMR 4, PMR5, PMR6, MLO, BIK1, DMR1, DMR6, DND1, CPR5, and SR1) were analyzed. Their genomic sequences were examined and SNPs/indels were annotated using the SNPeff pipeline. A total of 54,000 SNPs/indels were identified, among which 1300 were estimated to have a moderate impact (non-synonymous variants), while 120 were estimated to have a high impact (e.g., missense/nonsense/frameshift variants). The latter were then analyzed for their effect on gene functionality. A total of 103 genotypes showed one high-impact mutation in at least one of the scouted genes, while in 10 genotypes, more than 4 high-impact mutations in as many genes were detected. A set of 10 SNPs were validated through Sanger sequencing. Three genotypes carrying high-impact homozygous SNPs in S-genes were infected with Oidium neolycopersici, and two highlighted a significantly reduced susceptibility to the fungus. The existing mutations fall within the scope of a history of safe use and can be useful to guide risk assessment in evaluating the effect of new genomic techniques.

Diversity analyses in two ornamental and large-genome Ranunculaceae species based on a low-cost Klenow NGS-based protocol.

Persian buttercup (Ranunculus asiaticus L.) and poppy anemone (Anemone coronaria L.) are ornamental, outcrossing, perennial species belonging to the Ranunculaceae family, characterized by large and highly repetitive genomes. We applied K-seq protocol in both species to generate high-throughput sequencing data and produce a large number of genetic polymorphisms. The technique entails the application of Klenow polymerase-based PCR using short primers designed by analyzing k-mer sets in the genome sequence. To date the genome sequence of both species has not been released, thus we designed primer sets based on the reference the genome sequence of the related species Aquilegia oxysepala var. kansuensis (Brühl). A whole of 11,542 SNPs were selected for assessing genetic diversity of eighteen commercial varieties of R. asiaticus, while 1,752 SNPs for assessing genetic diversity in six cultivars of A. coronaria. UPGMA dendrograms were constructed and in R. asiaticus integrated in with PCA analysis. This study reports the first molecular fingerprinting within Persian buttercup, while the results obtained in poppy anemone were compared with a previously published SSR-based fingerprinting, proving K-seq to be an efficient protocol for the genotyping of complex genetic backgrounds.

CRISPR/Cas9-Based Knock-Out of the PMR4 Gene Reduces Susceptibility to Late Blight in Two Tomato Cultivars.

Phytophthora infestans, the causal agent of late blight (LB) in tomato (Solanum lycopersicum L.), is a devastating disease and a serious concern for plant productivity. The presence of susceptibility (S) genes in plants facilitates pathogen proliferation; thus, disabling these genes may help provide a broad-spectrum and durable type of tolerance/resistance. Previous studies on Arabidopsis and tomato have highlighted that knock-out mutants of the PMR4 susceptibility gene are tolerant to powdery mildew. Moreover, PMR4 knock-down in potato has been shown to confer tolerance to LB. To verify the same effect in tomato in the present study, a CRISPR-Cas9 vector containing four single guide RNAs (sgRNAs: sgRNA1, sgRNA6, sgRNA7, and sgRNA8), targeting as many SlPMR4 regions, was introduced via Agrobacterium-tumefaciens-mediated transformation into two widely grown Italian tomato cultivars: 'San Marzano' (SM) and 'Oxheart' (OX). Thirty-five plants (twenty-six SM and nine OX) were selected and screened to identify the CRISPR/Cas9-induced mutations. The different sgRNAs caused mutation frequencies ranging from 22.1 to 100% and alternatively precise insertions (sgRNA6) or deletions (sgRNA7, sgRNA1, and sgRNA8). Notably, sgRNA7 induced in seven SM genotypes a -7 bp deletion in the homozygous status, whereas sgRNA8 led to the production of fifteen SM genotypes with a biallelic mutation (-7 bp and -2 bp). Selected edited lines were inoculated with P. infestans, and four of them, fully knocked out at the PMR4 locus, showed reduced disease symptoms (reduction in susceptibility from 55 to 80%) compared to control plants. The four SM lines were sequenced using Illumina whole-genome sequencing for deeper characterization without exhibiting any evidence of mutations in the candidate off-target regions. Our results showed, for the first time, a reduced susceptibility to Phytophtora infestans in pmr4 tomato mutants confirming the role of KO PMR4 in providing broad-spectrum protection against pathogens.

First genetic maps development and QTL mining in Ranunculus asiaticus L. through ddRADseq.

Persian Buttercup (Ranunculus asiaticus L.; 2x=2n=16; estimated genome size: 7.6Gb) is an ornamental and perennial crop native of Asia Minor and Mediterranean basin, marketed both as cut flower or potted plant. Currently new varieties are developed by selecting plants carrying desirable traits in segregating progenies obtained by controlled mating, which are propagated through rhizomes or micro-propagated in vitro. In order to escalate selection efficiency and respond to market requests, more knowledge of buttercup genetics would facilitate the identification of markers associated with loci and genes controlling key ornamental traits, opening the way for molecular assisted breeding programs. Reduced-representation sequencing (RRS) represents a powerful tool for plant genotyping, especially in case of large genomes such as the one of buttercup, and have been applied for the development of high-density genetic maps in several species. We report on the development of the first molecular-genetic maps in R. asiaticus based on of a two-way pseudo-testcross strategy. A double digest restriction-site associated DNA (ddRAD) approach was applied for genotyping two F1 mapping populations, whose female parents were a genotype of a so called 'ponpon' and of a 'double flower' varieties, while the common male parental ('Cipro') was a genotype producing a simple flower. The ddRAD generated a total of ~2Gb demultiplexed reads, resulting in an average of 8,3M reads per line. The sstacks pipeline was applied for the construction of a mock reference genome based on sequencing data, and SNP markers segregating in only one of the parents were retained for map construction by treating the F1 population as a backcross. The four parental maps (two of the female parents and two of the common male parent) were aligned with 106 common markers and 8 linkage groups were identified, corresponding to the haploid chromosome number of the species. An average of 586 markers were associated with each parental map, with a marker density ranging from 1 marker/cM to 4.4 markers/cM. The developed maps were used for QTL analysis for flower color, leading to the identification of major QTLs for purple pigmentation. These results contribute to dissect on the genetics of Persian buttercup, enabling the development of new approaches for future varietal development.

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.

The Population Structure of a Globe Artichoke Worldwide Collection, as Revealed by Molecular and Phenotypic Analyzes.

The knowledge of the organization of the domesticated gene pool of crop species is an essential requirement to understand crop evolution, to rationalize conservation programs, and to support practical decisions in plant breeding. Here, we integrate simple sequence repeat (SSR) analysis and phenotypic characterization to investigate a globe artichoke collection that comprises most of the varieties cultivated worldwide. We show that the cultivated gene pool of globe artichoke includes five distinct genetic groups associated with the major phenotypic typologies: Catanesi (which based on our analysis corresponds to Violetti di Provenza), Spinosi, Violetti di Toscana, Romaneschi, and Macau. We observed that 17 and 11% of the molecular and phenotypic variance, respectively, is between these groups, while within groups, strong linkage disequilibrium and heterozygote excess are evident. The divergence between groups for quantitative traits correlates with the average broad-sense heritability within the groups. The phenotypic divergence between groups for both qualitative and quantitative traits is strongly and positively correlated with SSR divergence (FST) between groups. All this implies a low population size and strong bottleneck effects, and indicates a long history of clonal propagation and selection during the evolution of the domesticated gene pool of globe artichoke. Moreover, the comparison between molecular and phenotypic population structures suggests that harvest time, plant architecture (i.e., plant height, stem length), leaf spininess, head morphology (i.e., head shape, bract shape, spininess) together with the number of heads per plant were the main targets of selection during the evolution of the cultivated germplasm. We emphasize our findings in light of the potential exploitation of this collection for association mapping studies.

Genome-Wide Survey and Development of the First Microsatellite Markers Database (AnCorDB) in Anemone coronaria L.

Anemone coronaria L. (2n = 2x = 16) is a perennial, allogamous, highly heterozygous plant marketed as a cut flower or in gardens. Due to its large genome size, limited efforts have been made in order to develop species-specific molecular markers. We obtained the first draft genome of the species by Illumina sequencing an androgenetic haploid plant of the commercial line "MISTRAL® Magenta". The genome assembly was obtained by applying the MEGAHIT pipeline and consisted of 2 × 106 scaffolds. The SciRoKo SSR (Simple Sequence Repeats)-search module identified 401.822 perfect and 188.987 imperfect microsatellites motifs. Following, we developed a user-friendly "Anemone coronaria Microsatellite DataBase" (AnCorDB), which incorporates the Primer3 script, making it possible to design couples of primers for downstream application of the identified SSR markers. Eight genotypes belonging to eight cultivars were used to validate 62 SSRs and a subset of markers was applied for fingerprinting each cultivar, as well as to assess their intra-cultivar variability. The newly developed microsatellite markers will find application in Breeding Rights disputes, developing genetic maps, marker assisted breeding (MAS) strategies, as well as phylogenetic studies.

Transcriptome-Based Identification and Functional Characterization of NAC Transcription Factors Responsive to Drought Stress in Capsicum annuum L.

Capsicum annuum L. is one of the most cultivated Solanaceae species, and in the open field, water limitation leading to drought stress affects its fruit quality, fruit setting, fruit size and ultimately yield. We identified stage-specific and a common core set of differentially expressed genes, following RNA-seq transcriptome analyses of a breeding line subjected to acute drought stress followed by recovery (rewatering), at three stages of plant development. Among them, two NAC transcription factor (TF) genes, i.e., CaNAC072 and CaNAC104, were always upregulated after drought stress and downregulated after recovery. The two TF proteins were observed to be localized in the nucleus following their transient expression in Nicotiana benthamiana leaves. The expression of the two NACs was also induced by NaCl, polyethylene glycol (PEG) and abscisic acid (ABA) treatments, suggesting that CaNAC072 is an early, while CaNAC104 is a late abiotic stress-responsive gene. Virus-induced gene silencing (VIGS) of CaNAC104 did not affect the pepper plantlet's tolerance to drought stress, while VIGS of CaNAC072 increased drought tolerance. Heterologous expression of CaNAC072 in Arabidopsis thaliana as well as in plants mutated for its homolog ANAC072 did not increase drought stress tolerance. This highlights a different role of the two NAC homologs in the two species. Here, we discuss the complex role of NACs as transcriptional switches in the response to drought stress in bell pepper.

Global range expansion history of pepper (Capsicum spp.) revealed by over 10,000 genebank accessions.

Genebanks collect and preserve vast collections of plants and detailed passport information, with the aim of preserving genetic diversity for conservation and breeding. Genetic characterization of such collections has the potential to elucidate the genetic histories of important crops, use marker-trait associations to identify loci controlling traits of interest, search for loci undergoing selection, and contribute to genebank management by identifying taxonomic misassignments and duplicates. We conducted a genomic scan with genotyping by sequencing (GBS) derived single nucleotide polymorphisms (SNPs) of 10,038 pepper (Capsicum spp.) accessions from worldwide genebanks and investigated the recent history of this iconic staple. Genomic data detected up to 1,618 duplicate accessions within and between genebanks and showed that taxonomic ambiguity and misclassification often involve interspecific hybrids that are difficult to classify morphologically. We deeply interrogated the genetic diversity of the commonly consumed Capsicum annuum to investigate its history, finding that the kinds of peppers collected in broad regions across the globe overlap considerably. The method ReMIXTURE-using genetic data to quantify the similarity between the complement of peppers from a focal region and those from other regions-was developed to supplement traditional population genetic analyses. The results reflect a vision of pepper as a highly desirable and tradable cultural commodity, spreading rapidly throughout the globe along major maritime and terrestrial trade routes. Marker associations and possible selective sweeps affecting traits such as pungency were observed, and these traits were shown to be distributed nonuniformly across the globe, suggesting that human preferences exerted a primary influence over domesticated pepper genetic structure.

Oleosin Cor a 15 is a novel allergen for Italian hazelnut allergic children.

BACKGROUND: Hazelnut allergy, which is characterized by symptoms that range from mild to severe, is one of the most common allergies in children throughout Europe, and an accurate diagnosis of this allergy is therefore essential. However, lipophilic allergens, such as oleosins, are generally underrepresented in diagnostic tests. We therefore sought to characterize the IgE reactivity of raw and roasted hazelnut oleosins, using the sera of hazelnut-allergic pediatric patients. METHODS: Raw and roasted hazelnut oil body-associated proteins were analyzed by means of 1D and 2D electrophoresis and MS. Oleosin IgE reactivity was assessed by immunoblotting with the sera of 27 children who have confirmed hazelnut allergies and from 10 tolerant subjects. A molecular characterization of the oleosins was performed by interrogating the C. avellana cv. Jefferson and cv. TGL genomes, and through expression and purification of the recombinant new allergen. RESULTS: A proteomic and genomic investigation allowed two new oleosins to be identified, in addition to Cor a 12 and Cor a 13, in hazelnut oil bodies. One of the new oleosins was registered as a new allergen, according to the WHO/IUIS Allergen Nomenclature Subcommittee criteria, and termed Cor a 15. Cor a 15 was the most frequently immunorecognized oleosin in our cohort. Oleosins resulted to be the only immunorecognized allergens in a subgroup of allergic patients who showed low ImmunoCAP assay IgE values and positive OFC and PbP. Hazelnut roasting resulted in an increase in oleosin immunoreactivity. CONCLUSION: A novel hazelnut oleosin, named Cor a 15, has been discovered. Cor a 15 could play a role in eliciting an allergic reaction in a subgroup of pediatric patients that exclusively immunorecognize oleosins. The high prevalence of hazelnut oleosin sensitization here reported further confirms the need to include oleosins in routine diagnostic procedures.

Whole-genome assembly of Corylus avellana cv "Tonda Gentile delle Langhe" using linked-reads (10X Genomics).

The European hazelnut (Corylus avellana L.; 2n = 2x = 22) is a worldwide economically important tree nut that is cross-pollinated due to sporophytic incompatibility. Therefore, any individual plant is highly heterozygous. Cultivars are clonally propagated using mound layering, rooted suckers, and micropropagation. In recent years, the interest in this crop has increased, due to a growing demand related to the recognized health benefits of nut consumption. C. avellana cv "Tonda Gentile delle Langhe" ("TGdL") is well-known for its high kernel quality, and the premium price paid for this cultivar is an economic benefit for producers in northern Italy. Assembly of a high-quality genome is a difficult task in many plant species because of the high level of heterozygosity. We assembled a chromosome-level genome sequence of "TGdL" with a two-step approach. First, 10X Genomics Chromium Technology was used to create a high-quality sequence, which was then assembled into scaffolds with cv "Tombul" genome as the reference. Eleven pseudomolecules were obtained, corresponding to 11 chromosomes. A total of 11,046 scaffolds remained unplaced, representing 11% of the genome (46,504,161 bp). Gene prediction, performed with Maker-P software, identified 27,791 genes (AED ≤0.4 and 92% of BUSCO completeness), whose function was analyzed with BlastP and InterProScan software. To characterize "TGdL" specific genetic mechanisms, Orthofinder was used to detect orthologs between hazelnut and closely related species. The "TGdL" genome sequence is expected to be a powerful tool to understand hazelnut genetics and allow detection of markers/genes for important traits to be used in targeted breeding programs.

Mapping the Genetic Regions Responsible for Key Phenology-Related Traits in the European Hazelnut.

An increasing interest in the cultivation of (European) hazelnut (Corylus avellana) is driving a demand to breed cultivars adapted to non-conventional environments, particularly in the context of incipient climate change. Given that plant phenology is so strongly determined by genotype, a rational approach to support these breeding efforts will be to identify quantitative trait loci (QTLs) and the genes underlying the basis for adaptation. The present study was designed to map QTLs for phenology-related traits, such as the timing of both male and female flowering, dichogamy, and the period required for nuts to reach maturity. The analysis took advantage of an existing linkage map developed from a population of F1 progeny bred from the cross "Tonda Gentile delle Langhe" × "Merveille de Bollwiller," consisting in 11 LG. A total of 42 QTL-harboring regions were identified. Overall, 71 QTLs were detected, 49 on the TGdL map and 22 on the MB map; among these, 21 were classified as major; 13 were detected in at least two of the seasons (stable-major QTL). In detail, 20 QTLs were identified as contributing to the time of male flowering, 15 to time of female flowering, 25 to dichogamy, and 11 to time of nut maturity. LG02 was found to harbor 16 QTLs, while 15 QTLs mapped to LG10 and 14 to LG03. Many of the QTLs were clustered with one another. The major cluster was located on TGdL_02 and consisted of mainly major QTLs governing all the analyzed traits. A search of the key genomic regions revealed 22 candidate genes underlying the set of traits being investigated. Many of them have been described in the literature as involved in processes related to flowering, control of dormancy, budburst, the switch from vegetative to reproductive growth, or the morphogenesis of flowers and seeds.

Simultaneous CRISPR/Cas9 Editing of Three PPO Genes Reduces Fruit Flesh Browning in Solanum melongena L.

Polyphenol oxidases (PPOs) catalyze the oxidization of polyphenols, which in turn causes the browning of the eggplant berry flesh after cutting. This has a negative impact on fruit quality for both industrial transformation and fresh consumption. Ten PPO genes (named SmelPPO1-10) were identified in eggplant thanks to the recent availability of a high-quality genome sequence. A CRISPR/Cas9-based mutagenesis approach was applied to knock-out three target PPO genes (SmelPPO4, SmelPPO5, and SmelPPO6), which showed high transcript levels in the fruit after cutting. An optimized transformation protocol for eggplant cotyledons was used to obtain plants in which Cas9 is directed to a conserved region shared by the three PPO genes. The successful editing of the SmelPPO4, SmelPPO5, and SmelPPO6 loci of in vitro regenerated plantlets was confirmed by Illumina deep sequencing of amplicons of the target sites. Besides, deep sequencing of amplicons of the potential off-target loci identified in silico proved the absence of detectable non-specific mutations. The induced mutations were stably inherited in the T1 and T2 progeny and were associated with a reduced PPO activity and browning of the berry flesh after cutting. Our results provide the first example of the use of the CRISPR/Cas9 system in eggplant for biotechnological applications and open the way to the development of eggplant genotypes with low flesh browning which maintain a high polyphenol content in the berries.

Transcriptome Analyses and Antioxidant Activity Profiling Reveal the Role of a Lignin-Derived Biostimulant Seed Treatment in Enhancing Heat Stress Tolerance in Soybean.

Soybean (Glycine max Merr.) is a worldwide important legume crop, whose growth and yield are negatively affected by heat stress at germination time. Here, we tested the role of a biostimulant based on lignin derivatives, plant-derived amino acids, and molybdenum in enhancing soybean heat stress tolerance when applied on seeds. After treatment with the biostimulant at 35 °C, the seed biometric parameters were positively influenced after 24 h, meanwhile, germination percentage was increased after 72 h (+10%). RNA-Seq analyses revealed a modulation of 879 genes (51 upregulated and 828 downregulated) in biostimulant-treated seeds as compared with the control, at 24 h after incubation at 35 °C. Surprisingly, more than 33% of upregulated genes encoded for ribosomal RNA (rRNA) methyltransferases and proteins involved in the ribosome assembly, acting in a specific protein network. Conversely, the downregulated genes were involved in stress response, hormone signaling, and primary metabolism. Finally, from a biochemical point of view, the dramatic H2O2 reduction 40%) correlated to a strong increase in non-protein thiols (+150%), suggested a lower oxidative stress level in biostimulant-treated seeds, at 24 h after incubation at 35 °C. Our results provide insights on the biostimulant mechanism of action and on its application for seed treatments to improve heat stress tolerance during germination.

"Mind the Gap": Hi-C Technology Boosts Contiguity of the Globe Artichoke Genome in Low-Recombination Regions.

Globe artichoke (Cynara cardunculus var. scolymus; 2n2x=34) is cropped largely in the Mediterranean region, being Italy the leading world producer; however, over time, its cultivation has spread to the Americas and China. In 2016, we released the first (v1.0) globe artichoke genome sequence (http://www.artichokegenome.unito.it/). Its assembly was generated using ∼133-fold Illumina sequencing data, covering 725 of the 1,084 Mb genome, of which 526 Mb (73%) were anchored to 17 chromosomal pseudomolecules. Based on v1.0 sequencing data, we generated a new genome assembly (v2.0), obtained from a Hi-C (Dovetail) genomic library, and which improves the scaffold N50 from 126 kb to 44.8 Mb (∼356-fold increase) and N90 from 29 kb to 17.8 Mb (∼685-fold increase). While the L90 of the v1.0 sequence included 6,123 scaffolds, the new v2.0 just 15 super-scaffolds, a number close to the haploid chromosome number of the species. The newly generated super-scaffolds were assigned to pseudomolecules using reciprocal blast procedures. The cumulative size of unplaced scaffolds in v2.0 was reduced of 165 Mb, increasing to 94% the anchored genome sequence. The marked improvement is mainly attributable to the ability of the proximity ligation-based approach to deal with both heterochromatic (e.g.: peri-centromeric) and euchromatic regions during the assembly procedure, which allowed to physically locate low recombination regions. The new high-quality reference genome enhances the taxonomic breadth of the data available for comparative plant genomics and led to a new accurate gene prediction (28,632 genes), thus promoting the map-based cloning of economically important genes.

Development of High-Density Genetic Linkage Maps and Identification of Loci for Chestnut Gall Wasp Resistance in Castanea spp.

Castanea sativa is an important multipurpose species in Europe for nut and timber production as well as for its role in the landscape and in the forest ecosystem. This species has low tolerance to chestnut gall wasp (Dryocosmus kuriphilus Yasumatsu), which is a pest that was accidentally introduced into Europe in early 2000 and devastated forest and orchard trees. Resistance to the gall wasp was found in the hybrid cultivar 'Bouche de Bétizac' (C. sativa × C. crenata) and studied by developing genetic linkage maps using a population derived from a cross between 'Bouche de Bétizac' and the susceptible cultivar 'Madonna' (C. sativa). The high-density genetic maps were constructed using double-digest restriction site-associated DNA-seq and simple sequence repeat markers. The map of 'Bouche de Bétizac' consisted of 1459 loci and spanned 809.6 cM; the map of 'Madonna' consisted of 1089 loci and spanned 753.3 cM. In both maps, 12 linkage groups were identified. A single major QTL was recognized on the 'Bouche de Bétizac' map, explaining up to 67-69% of the phenotypic variance of the resistance trait (Rdk1). The Rdk1 quantitative trait loci (QTL) region included 11 scaffolds and two candidate genes putatively involved in the resistance response were identified. This study will contribute to C. sativa breeding programs and to the study of Rdk1 genes.