Analysis of >3400 worldwide eggplant accessions reveals two independent domestication events and multiple migration-diversification routes.

Eggplant (Solanum melongena) is an important Solanaceous crop, widely cultivated and consumed in Asia, the Mediterranean basin, and Southeast Europe. Its domestication centers and migration and diversification routes are still a matter of debate. We report the largest georeferenced and genotyped collection to this date for eggplant and its wild relatives, consisting of 3499 accessions from seven worldwide genebanks, originating from 105 countries in five continents. The combination of genotypic and passport data points to the existence of at least two main centers of domestication, in Southeast Asia and the Indian subcontinent, with limited genetic exchange between them. The wild and weedy eggplant ancestor S. insanum shows admixture with domesticated S. melongena, similar to what was described for other fruit-bearing Solanaceous crops such as tomato and pepper and their wild ancestors. After domestication, migration and admixture of eggplant populations from different regions have been less conspicuous with respect to tomato and pepper, thus better preserving 'local' phenotypic characteristics. The data allowed the identification of misclassified and putatively duplicated accessions, facilitating genebank management. All the genetic, phenotypic, and passport data have been deposited in the Open Access G2P-SOL database, and constitute an invaluable resource for understanding the domestication, migration and diversification of this cosmopolitan vegetable.

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.

Conventional and new genetic resources for an eggplant breeding revolution.

Eggplant (Solanum melongena) is a major vegetable crop with great potential for genetic improvement owing to its large and mostly untapped genetic diversity. It is closely related to over 500 species of Solanum subgenus Leptostemonum that belong to its primary, secondary, and tertiary genepools and exhibit a wide range of characteristics useful for eggplant breeding, including traits adaptive to climate change. Germplasm banks worldwide hold more than 19 000 accessions of eggplant and related species, most of which have yet to be evaluated. Nonetheless, eggplant breeding using the cultivated S. melongena genepool has yielded significantly improved varieties. To overcome current breeding challenges and for adaptation to climate change, a qualitative leap forward in eggplant breeding is necessary. The initial findings from introgression breeding in eggplant indicate that unleashing the diversity present in its relatives can greatly contribute to eggplant breeding. The recent creation of new genetic resources such as mutant libraries, core collections, recombinant inbred lines, and sets of introgression lines will be another crucial element and will require the support of new genomics tools and biotechnological developments. The systematic utilization of eggplant genetic resources supported by international initiatives will be critical for a much-needed eggplant breeding revolution to address the challenges posed by climate change.

Improved genome assembly and pan-genome provide key insights into eggplant domestication and breeding.

Eggplant (Solanum melongena L.) is an important horticultural crop and one of the most widely grown vegetables from the Solanaceae family. It was domesticated from a wild, prickly progenitor carrying small, round, non-anthocyanic fruits. We obtained a novel, highly contiguous genome assembly of the eggplant '67/3' reference line, by Hi-C retrofitting of a previously released short read- and optical mapping-based assembly. The sizes of the 12 chromosomes and the fraction of anchored genes in the improved assembly were comparable to those of a chromosome-level assembly. We resequenced 23 accessions of S. melongena representative of the worldwide phenotypic, geographic, and genetic diversity of the species, and one each from the closely related species Solanum insanum and Solanum incanum. The eggplant pan-genome contained approximately 51.5 additional megabases and 816 additional genes compared with the reference genome, while the pan-plastome showed little genetic variation. We identified 53 selective sweeps related to fruit color, prickliness, and fruit shape in the nuclear genome, highlighting selection leading to the emergence of present-day S. melongena cultivars from its wild ancestors. Candidate genes underlying the selective sweeps included a MYBL1 repressor and CHALCONE ISOMERASE (for fruit color), homologs of Arabidopsis GLABRA1 and GLABROUS INFLORESCENCE STEMS2 (for prickliness), and orthologs of tomato FW2.2, OVATE, LOCULE NUMBER/WUSCHEL, SUPPRESSOR OF OVATE, and CELL SIZE REGULATOR (for fruit size/shape), further suggesting that selection for the latter trait relied on a common set of orthologous genes in tomato and eggplant.

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.

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.

The influence of acute water stresses on the biochemical composition of bell pepper (Capsicum annuum L.) berries.

BACKGROUND: Crops are exposed to recurrent and acute drought stress episodes during their vegetative and reproductive cycles, and these episodes are increasingly frequent due to ongoing climate change. Sweet pepper (Capsicum annuum), alias bell pepper, is one of the most widely cultivated vegetables and is grown in open fields worldwide. Here we assessed the effect of acute water stress, applied to a breeding line of sweet pepper at three stages of plant development: five true-leaves (Stage 1), production of the third flower (Stage 2) and setting of the first fruit (Stage 3), on the production and biochemical composition of its ripe fruits. RESULTS: The water stress at Stages 1 and 2 induced a delay in fruit ripening, while at Stage 3 caused a drop in production. The biochemical composition of ripe fruits was assessed by quantifying their content in vitamin C, sugars, organic acids, flavonoids as well as 190 volatile organic compounds, mainly belonging to the chemical classes of hydrocarbons, alcohols, ketones, esters, terpenes, aldehydes and ethers. Our results highlight that, at different stages of plant development, acute water stresses modulate differently the accumulation of bioactive compounds in fruits, which play a key role in setting the redox-status and osmotic adjustment of the plant. This was also the case for volatile compounds since, within each chemical class, different compounds varied their content in ripe fruits. CONCLUSIONS: On the whole, our results demonstrate that water stresses potentially affect the organoleptic and sensory qualities of bell pepper fruits depending on when they occur. © 2021 Society of Chemical Industry.

Dual catalytic activity of hydroxycinnamoyl-coenzyme A quinate transferase from tomato allows it to moonlight in the synthesis of both mono- and dicaffeoylquinic acids.

Tomato (Solanum lycopersicum), like other Solanaceous species, accumulates high levels of antioxidant caffeoylquinic acids, which are strong bioactive molecules and protect plants against biotic and abiotic stresses. Among these compounds, the monocaffeoylquinic acids (e.g. chlorogenic acid [CGA]) and the dicaffeoylquinic acids (diCQAs) have been found to possess marked antioxidative properties. Thus, they are of therapeutic interest both as phytonutrients in foods and as pharmaceuticals. Strategies to increase diCQA content in plants have been hampered by the modest understanding of their biosynthesis and whether the same pathway exists in different plant species. Incubation of CGA with crude extracts of tomato fruits led to the formation of two new products, which were identified by liquid chromatography-mass spectrometry as diCQAs. This chlorogenate:chlorogenate transferase activity was partially purified from ripe fruit. The final protein fraction resulted in 388-fold enrichment of activity and was subjected to trypsin digestion and mass spectrometric sequencing: a hydroxycinnamoyl-Coenzyme A:quinate hydroxycinnamoyl transferase (HQT) was selected as a candidate protein. Assay of recombinant HQT protein expressed in Escherichia coli confirmed its ability to synthesize diCQAs in vitro. This second activity (chlorogenate:chlorogenate transferase) of HQT had a low pH optimum and a high Km for its substrate, CGA. High concentrations of CGA and relatively low pH occur in the vacuoles of plant cells. Transient assays demonstrated that tomato HQT localizes to the vacuole as well as to the cytoplasm of plant cells, supporting the idea that in this species, the enzyme catalyzes different reactions in two subcellular compartments.

Linkage disequilibrium and genome-wide association analysis for anthocyanin pigmentation and fruit color in eggplant.

BACKGROUND: The genome-wide association (GWA) approach represents an alternative to biparental linkage mapping for determining the genetic basis of trait variation. Both approaches rely on recombination to re-arrange the genome, and seek to establish correlations between phenotype and genotype. The major advantages of GWA lie in being able to sample a much wider range of the phenotypic and genotypic variation present, in being able to exploit multiple rounds of historical recombination in many different lineages and to include multiple accessions of direct relevance to crop improvement. RESULTS: A 191 accessions eggplant (Solanum melongena L.) association panel, comprising a mixture of breeding lines, old varieties and landrace selections originating from Asia and the Mediterranean Basin, was SNP genotyped and scored for anthocyanin pigmentation and fruit color at two locations over two years. The panel formed two major clusters, reflecting geographical provenance and fruit type. The global level of linkage disequilibrium was 3.4 cM. A mixed linear model appeared to be the most appropriate for GWA. A set of 56 SNP locus/phenotype associations was identified and the genomic regions harboring these loci were distributed over nine of the 12 eggplant chromosomes. The associations were compared with the location of known QTL for the same traits. CONCLUSION: The GWA mapping approach was effective in validating a number of established QTL and, thanks to the wide diversity captured by the panel, was able to detect a series of novel marker/trait associations.

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.

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.

2-D DIGE analysis of UV-C radiation-responsive proteins in globe artichoke leaves.

Plants respond to ultraviolet stress inducing a self-defence through the regulation of specific gene family members. The UV acclimation is the result of biochemical and physiological processes, such as enhancement of the antioxidant enzymatic system and accumulation of UV-absorbing phenolic compounds (e.g. flavonoids). Globe artichoke is an attractive species for studying the protein network involved in UV stress response, being characterized by remarkable levels of inducible antioxidants. Proteomic tools can assist the evaluation of the expression patterns of UV-responsive proteins and we applied the difference in-gel electrophoresis (DIGE) technology for monitoring the globe artichoke proteome variation at four time points following an acute UV-C exposure. A total of 145 UV-C-modulated proteins were observed and 119 were identified by LC-MS/MS using a ∼144,000 customized Compositae protein database, which included about 19,000 globe artichoke unigenes. Proteins were Gene Ontology (GO) categorized, visualized on their pathways and their behaviour was discussed. A predicted protein interaction network was produced and highly connected hub-like proteins were highlighted. Most of the proteins differentially modulated were chloroplast located, involved in photosynthesis, sugar metabolisms, protein folding and abiotic stress. The identification of UV-C-responsive proteins may contribute to shed light on the molecular mechanisms underlying plant responses to UV stress.

RAD tag sequencing as a source of SNP markers in Cynara cardunculus L.

BACKGROUND: The globe artichoke (Cynara cardunculus L. var. scolymus) genome is relatively poorly explored, especially compared to those of the other major Asteraceae crops sunflower and lettuce. No SNP markers are in the public domain. We have combined the recently developed restriction-site associated DNA (RAD) approach with the Illumina DNA sequencing platform to effect the rapid and mass discovery of SNP markers for C. cardunculus. RESULTS: RAD tags were sequenced from the genomic DNA of three C. cardunculus mapping population parents, generating 9.7 million reads, corresponding to ~1 Gbp of sequence. An assembly based on paired ends produced ~6.0 Mbp of genomic sequence, separated into ~19,000 contigs (mean length 312 bp), of which ~21% were fragments of putative coding sequence. The shared sequences allowed for the discovery of ~34,000 SNPs and nearly 800 indels, equivalent to a SNP frequency of 5.6 per 1,000 nt, and an indel frequency of 0.2 per 1,000 nt. A sample of heterozygous SNP loci was mapped by CAPS assays and this exercise provided validation of our mining criteria. The repetitive fraction of the genome had a high representation of retrotransposon sequence, followed by simple repeats, AT-low complexity regions and mobile DNA elements. The genomic k-mers distribution and CpG rate of C. cardunculus, compared with data derived from three whole genome-sequenced dicots species, provided a further evidence of the random representation of the C. cardunculus genome generated by RAD sampling. CONCLUSION: The RAD tag sequencing approach is a cost-effective and rapid method to develop SNP markers in a highly heterozygous species. Our approach permitted to generate a large and robust SNP datasets by the adoption of optimized filtering criteria.

Large-scale transcriptome characterization and mass discovery of SNPs in globe artichoke and its related taxa.

Cynara cardunculus (2n = 2× = 34) is a member of the Asteraceae family that contributes significantly to the agricultural economy of the Mediterranean basin. The species includes two cultivated varieties, globe artichoke and cardoon, which are grown mainly for food. Cynara cardunculus is an orphan crop species whose genome/transcriptome has been relatively unexplored, especially in comparison to other Asteraceae crops. Hence, there is a significant need to improve its genomic resources through the identification of novel genes and sequence-based markers, to design new breeding schemes aimed at increasing quality and crop productivity. We report the outcome of cDNA sequencing and assembly for eleven accessions of C. cardunculus. Sequencing of three mapping parental genotypes using Roche 454-Titanium technology generated 1.7 × 106 reads, which were assembled into 38,726 reference transcripts covering 32 Mbp. Putative enzyme-encoding genes were annotated using the KEGG-database. Transcription factors and candidate resistance genes were surveyed as well. Paired-end sequencing was done for cDNA libraries of eight other representative C. cardunculus accessions on an Illumina Genome Analyzer IIx, generating 46 × 106 reads. Alignment of the IGA and 454 reads to reference transcripts led to the identification of 195,400 SNPs with a Bayesian probability exceeding 95%; a validation rate of 90% was obtained by Sanger-sequencing of a subset of contigs. These results demonstrate that the integration of data from different NGS platforms enables large-scale transcriptome characterization, along with massive SNP discovery. This information will contribute to the dissection of key agricultural traits in C. cardunculus and facilitate the implementation of marker-assisted selection programs.

Identifying Quantitative Trait Loci for Thousand Grain Weight in Eggplant by Genome Re-Sequencing Analysis.

Eggplant (Solanum melongena L.; 2n = 24) is one of the most important Solanaceae vegetables and is primarily cultivated in China (approximately 42% of world production) and India (approximately 39%). Thousand-grain weight (TGW) is an important trait that affects eggplant breeding cost and variety promotion. This trait is controlled by quantitative trait loci (QTLs); however, no quantitative trait loci (QTL) has been reported for TGW in eggplant so far, and its potential genetic basis remain unclear. In this study, two eggplant lines, 17C01 (P1, wild resource, small seed) and 17C02 (P2, cultivar, large seed), were crossed to develop F1, F2 (308 lines), BC1P1 (44 lines), and BC1P2 (44 lines) populations for quantitative trait association analysis. The TGWs of P1, P2 and F1 were determined as 3.00, 3.98 and 3.77 g, respectively. The PG-ADI (polygene-controlled additive-dominance-epistasis) genetic model was identified as the optimal model for TGW and the polygene heritability value in the F2 generation was as high as 80.87%. A high-quality genetic linkage bin map was constructed with resequencing analysis. The map contained 3,918 recombination bins on 12 chromosomes, and the total length was 1,384.62 cM. A major QTL (named as TGW9.1) located on chromosome 9 was identified to be strongly associated with eggplant TGW, with a phenotypic variance explanation of 20.51%. A total of 45 annotated genes were identified in the genetic region of TGW9.1. Based on the annotation of Eggplant genome V3 and orthologous genes in Arabidopsis thaliana, one candidate gene SMEL_009g329850 (SmGTS1, encoding a putative ubiquitin ligase) contains 4 SNPs and 2 Indels consecutive intron mutations in the flank of the same exon in P1. SmGTS1 displayed significantly higher expression in P1 and was selected as a potential candidate gene controlling TGW in eggplant. The present results contribute to shed light on the genetic basis of the traits exploitable in future eggplant marker-assisted selection (MAS) breeding.

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.

Identification of SNP and SSR markers in eggplant using RAD tag sequencing.

BACKGROUND: The eggplant (Solanum melongena L.) genome is relatively unexplored, especially compared to those of the other major Solanaceae crops tomato and potato. In particular, no SNP markers are publicly available; on the other hand, over 1,000 SSR markers were developed and publicly available. We have combined the recently developed Restriction-site Associated DNA (RAD) approach with Illumina DNA sequencing for rapid and mass discovery of both SNP and SSR markers for eggplant. RESULTS: RAD tags were generated from the genomic DNA of a pair of eggplant mapping parents, and sequenced to produce ~17.5 Mb of sequences arrangeable into ~78,000 contigs. The resulting non-redundant genomic sequence dataset consisted of ~45,000 sequences, of which ~29% were putative coding sequences and ~70% were in common between the mapping parents. The shared sequences allowed the discovery of ~10,000 SNPs and nearly 1,000 indels, equivalent to a SNP frequency of 0.8 per Kb and an indel frequency of 0.07 per Kb. Over 2,000 of the SNPs are likely to be mappable via the Illumina GoldenGate assay. A subset of 384 SNPs was used to successfully fingerprint a panel of eggplant germplasm, producing a set of informative diversity data. The RAD sequences also included nearly 2,000 putative SSRs, and primer pairs were designed to amplify 1,155 loci. CONCLUSION: The high throughput sequencing of the RAD tags allowed the discovery of a large number of DNA markers, which will prove useful for extending our current knowledge of the genome organization of eggplant, for assisting in marker-aided selection and for carrying out comparative genomic analyses within the Solanaceae family.

An Eggplant Recombinant Inbred Population Allows the Discovery of Metabolic QTLs Controlling Fruit Nutritional Quality.

Eggplant (Solanum melongena L.) represents the third most important crop of the Solanaceae family and is an important component of our daily diet. A population of 164 F6 recombinant inbred lines (RILs), derived from two eggplant lines differing with respect to several key agronomic traits, "305E40" and "67/3," was grown to the commercial maturation stage, and fruits were harvested, separated into peel and flesh, and subjected to liquid chromatography Liquid Chromatography/Mass Spectrometry (LC/MS) analysis. Through a combination of untargeted and targeted metabolomics approaches, a number of metabolites belonging to the glycoalkaloid, anthocyanin, and polyamine classes and showing a differential accumulation in the two parental lines and F1 hybrid were identified. Through metabolic profiling of the RILs, we identified several metabolomic quantitative trait loci (mQTLs) associated with the accumulation of those metabolites. Each of the metabolic traits proved to be controlled by one or more quantitative trait loci (QTLs); for most of the traits, one major mQTL (phenotypic variation explained [PVE] ≥ 10%) was identified. Data on mQTL mapping and dominance-recessivity relationships of measured compounds in the parental lines and F1 hybrid, as well as an analysis of the candidate genes underlying the QTLs and of their sequence differences in the two parental lines, suggested a series of candidate genes underlying the traits under study.