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.

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.

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.

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.

A Compendium for Novel Marker-Based Breeding Strategies in Eggplant.

The worldwide production of eggplant is estimated at about 58 Mt, with China, India and Egypt being the major producing countries. Breeding efforts in the species have mainly focused on increasing productivity, abiotic and biotic tolerance/resistance, shelf-life, the content of health-promoting metabolites in the fruit rather than decreasing the content of anti-nutritional compounds in the fruit. From the literature, we collected information on mapping quantitative trait loci (QTLs) affecting eggplant's traits following a biparental or multi-parent approach as well as genome-wide association (GWA) studies. The positions of QTLs were lifted according to the eggplant reference line (v4.1) and more than 700 QTLs were identified, here organized into 180 quantitative genomic regions (QGRs). Our findings thus provide a tool to: (i) determine the best donor genotypes for specific traits; (ii) narrow down QTL regions affecting a trait by combining information from different populations; (iii) pinpoint potential candidate genes.

Mutations in the SmAPRR2 transcription factor suppressing chlorophyll pigmentation in the eggplant fruit peel are key drivers of a diversified colour palette.

Understanding the mechanisms by which chlorophylls are synthesized in the eggplant (Solanum melongena) fruit peel is of great relevance for eggplant breeding. A multi-parent advanced generation inter-cross (MAGIC) population and a germplasm collection have been screened for green pigmentation in the fruit peel and used to identify candidate genes for this trait. A genome-wide association study (GWAS) performed with 420 MAGIC individuals revealed a major association on chromosome 8 close to a gene similar to APRR2. Two variants in SmAPRR2, predicted as having a high impact effect, were associated with the absence of fruit chlorophyll pigmentation in the MAGIC population, and a large deletion of 5.27 kb was found in two reference genomes of accessions without chlorophyll in the fruit peel. The validation of the candidate gene SmAPRR2 was performed by its sequencing in a set of MAGIC individuals and through its de novo assembly in 277 accessions from the G2P-SOL eggplant core collection. Two additional mutations in SmAPRR2 associated with the lack of chlorophyll were identified in the core collection set. The phylogenetic analysis of APRR2 reveals orthology within Solanaceae and suggests that specialization of APRR2-like genes occurred independently in Cucurbitaceae and Solanaceae. A strong geographical differentiation was observed in the frequency of predominant mutations in SmAPRR2, resulting in a lack of fruit chlorophyll pigmentation and suggesting that this phenotype may have arisen and been selected independently several times. This study represents the first identification of a major gene for fruit chlorophyll pigmentation in the eggplant fruit.

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.

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.

Grafting vigour is associated with DNA de-methylation in eggplant.

In horticulture, grafting is a popular technique used to combine positive traits from two different plants. This is achieved by joining the plant top part (scion) onto a rootstock which contains the stem and roots. Rootstocks can provide resistance to stress and increase plant production, but despite their wide use, the biological mechanisms driving rootstock-induced alterations of the scion phenotype remain largely unknown. Given that epigenetics plays a relevant role during distance signalling in plants, we studied the genome-wide DNA methylation changes induced in eggplant (Solanum melongena) scion using two interspecific rootstocks to increase vigour. We found that vigour was associated with a change in scion gene expression and a genome-wide hypomethylation in the CHH context. Interestingly, this hypomethylation correlated with the downregulation of younger and potentially more active long terminal repeat retrotransposable elements (LTR-TEs), suggesting that graft-induced epigenetic modifications are associated with both physiological and molecular phenotypes in grafted plants. Our results indicate that the enhanced vigour induced by heterografting in eggplant is associated with epigenetic modifications, as also observed in some heterotic hybrids.

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.

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.

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.

The Genetic Basis of Tomato Aroma.

Tomato (Solanum lycopersicum L.) aroma is determined by the interaction of volatile compounds (VOCs) released by the tomato fruits with receptors in the nose, leading to a sensorial impression, such as "sweet", "smoky", or "fruity" aroma. Of the more than 400 VOCs released by tomato fruits, 21 have been reported as main contributors to the perceived tomato aroma. These VOCs can be grouped in five clusters, according to their biosynthetic origins. In the last decades, a vast array of scientific studies has investigated the genetic component of tomato aroma in modern tomato cultivars and their relatives. In this paper we aim to collect, compare, integrate and summarize the available literature on flavour-related QTLs in tomato. Three hundred and 5ifty nine (359) QTLs associated with tomato fruit VOCs were physically mapped on the genome and investigated for the presence of potential candidate genes. This review makes it possible to (i) pinpoint potential donors described in literature for specific traits, (ii) highlight important QTL regions by combining information from different populations, and (iii) pinpoint potential candidate genes. This overview aims to be a valuable resource for researchers aiming to elucidate the genetics underlying tomato flavour and for breeders who aim to improve tomato aroma.

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.

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.