Less is more: CRISPR/Cas9-based mutations in DND1 gene enhance tomato resistance to powdery mildew with low fitness costs.

Powdery mildew (PM), triggered by Oidium neolycopersici, represents a significant threat and a major concern for the productivity of tomato plants (Solanum lycopersicum L.). The presence of susceptibility (S) genes in plants facilitates pathogen proliferation and their dysfunction can lead to a recessively inherited broad-spectrum and durable type of resistance. Past studies have demonstrated that disrupting the function of DND1 (Defense No Death 1) increases plant resilience against various pathogens, such as powdery mildew (PM), but this comes at the cost of negatively affecting the overall health and vigor of the plant. To investigate the possibility of minimizing the adverse effects of the dnd1 mutation while boosting disease resistance, a CRISPR-Cas9 construct with four single guide RNAs targeting three exons of SlDND1 (Solyc02g088560.4.1) was designed and introduced into the tomato variety Moneymaker (MM) through Agrobacterium tumefaciens-mediated transformation. Three T1 lines (named E1, E3 and E4) were crossed with MM and then selfed to produce TF2 families. All the TF2 plants in homozygous state dnd1/dnd1, showed reduced PM symptoms compared to the heterozygous (DND1/dnd1) and wild type (DND1/DND1) ones. Two full knock-out (KO) mutant events (E1 and E4) encoding truncated DND1 proteins, exhibited clear dwarfness and auto-necrosis phenotypes, while mutant event E3 harbouring deletions of 3 amino acids, showed normal growth in height with less auto-necrotic spots. Analysis of the 3D structures of both the reference and the mutant proteins revealed significant conformational alterations in the protein derived from E3, potentially impacting its function. A dnd1/dnd1 TF2 line (TV181848-9, E3) underwent whole-genome sequencing using Illumina technology, which confirmed the absence of off-target mutations in selected genomic areas. Additionally, no traces of the Cas9 gene were detected, indicating its elimination through segregation. Our findings confirm the role of DND1 as an S-gene in tomato because impairment of this gene leads to a notable reduction in susceptibility to O. neolycopersici. Moreover, we provide, for the first time, a dnd1 mutant allele (E3) that exhibits fitness advantages in comparison with previously reported dnd1 mutant alleles, indicating a possible way to breed with dnd1 mutants.

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.

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.

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.

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.

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.

A New Intra-Specific and High-Resolution Genetic Map of Eggplant Based on a RIL Population, and Location of QTLs Related to Plant Anthocyanin Pigmentation and Seed Vigour.

Eggplant is the second most important solanaceous berry-producing crop after tomato. Despite mapping studies based on bi-parental progenies and GWAS approaches having been performed, an eggplant intraspecific high-resolution map is still lacking. We developed a RIL population from the intraspecific cross '305E40', (androgenetic introgressed line carrying the locus Rfo-Sa1 conferring Fusarium resistance) x '67/3' (breeding line whose genome sequence was recently released). One hundred and sixty-three RILs were genotyped by a genotype-by-sequencing (GBS) approach, which allowed us to identify 10,361 polymorphic sites. Overall, 267 Gb of sequencing data were generated and ~773 M Illumina paired end (PE) reads were mapped against the reference sequence. A new linkage map was developed, including 7249 SNPs assigned to the 12 chromosomes and spanning 2169.23 cM, with iaci@liberoan average distance of 0.4 cM between adjacent markers. This was used to elucidate the genetic bases of seven traits related to anthocyanin content in different organs recorded in three locations as well as seed vigor. Overall, from 7 to 17 QTLs (at least one major QTL) were identified for each trait. These results demonstrate that our newly developed map supplies valuable information for QTL fine mapping, candidate gene identification, and the development of molecular markers for marker assisted selection (MAS) of favorable alleles.

Diagnostic accuracy of fine-needle aspiration of solid pancreatic lesions guided by endoscopic ultrasound elastography.

BACKGROUND: Real-time elastography (RTE) may increase the diagnostic accuracy of fine-needle aspiration guided by endoscopic ultrasound. The aim of this study was to establish the diagnostic accuracy, sensitivity, and specificity of this combined methodological approach in a cohort of patients with solid pancreatic masses. METHODS: We reviewed data from 54 patients with solid pancreatic lesions referred to our institution between January 2014 and June 2015. RTE, assessed in terms of strain ratio, was performed both qualitatively and semi-quantitatively, and a 25G needle was inserted into the most suspicious part of the lesion. Sensitivity, specificity, diagnostic accuracy, positive and negative predictive values were calculated. RESULTS: The median lesion size was 35 mm (interquartile range: 25-43 mm). A diagnosis of adenocarcinoma was confirmed in 85.1% of cases. RTE, with a strain ratio cutoff of 4.21, showed a sensitivity of 86.9%, a specificity of 75%, and diagnostic accuracy of 85.1%. The diagnostic accuracy, sensitivity, and specificity of the combined methodology were 94.4%, 93.4%, and 100%, respectively. The positive predictive value was 100%, the negative predictive value 72.7% and the negative likelihood ratio 6.5. No severe adverse events were registered. CONCLUSION: The combination of RTE with endoscopic ultrasound-guided fine-needle aspiration appears to be an efficient and safe technique for the characterization of solid pancreatic masses.