Andrea Mazzucato

Tomato high pigment-2 (hp-2^dg, hp-2, and hp-2^j) mutant lines are characterized by mutations in the DE-ETIOLATED1 (SlDET1; Solyc01g056340) gene. SlDET1 is responsible for encoding a nuclear protein that acts as a negative regulator involved in light signaling, repressing photomorphogenesis. These tomato mutant lines are known for increased levels of antioxidant pigments in fruits, such as flavonoids and carotenoids, compared to the wild-type fruits. In this study, CRISPR/Cas9, followed by the non-homologous end joining mechanism of repair (NHEJ), was used to target the SlDET1 gene and investigate whether the effects of these mutations could mimic the effects of hp-2 mutant lines, improving the nutritional features of tomato fruits. Our results indicated that mutations generated by CRISPR/Cas9 NHEJ in the hp-2 and hp-2^j regions (exon 11) resulted in significant changes in the SlDET1 coding and protein sequences. These mutations caused a low survival rate of edited sprouts and regenerated plants with a very compromised capacity of allelic heritability of these mutations for the following generations. However, regenerated plants containing these site-specific mutations in the SlDET1 gene showed higher levels of phytochemicals in ripe fruits. Furthermore, these edited plants also showed an upregulation of structural genes involved in the synthesis of these biocompounds. Although the SlDET1 gene could be considered an interesting target gene for the nutritional improvement of tomato fruits, our results showed that mutations within its exon 11 are quite critical and can induce severe perturbations in plant physiology, with a compromised possibility to develop new stable edited lines.

The extensive diversification of flower shape and organs underpins the adaptive success of angiosperms. Despite substantial knowledge of the molecular mechanisms controlling flower induction and development, few studies have quantified the variability in floral traits within species or explored their correlation with other reproductive traits. In cultivated tomato (Solanum lycopersicum L.), human selection has driven fruit diversification in terms of size and shape. In the present study, 48 landraces representing tomato diversity in reproduction-related characteristics were phenotyped for 18 flower structural or dimensional traits. Flower traits exhibited lower coefficients of variation compared to other reproductive traits, though organ numbers showed high heritability values. Flower organ number and size were tightly correlated, but the correlation between dimensional traits was weaker. This likely reflects the selective pressures on pistil traits during domestication, including specific mutations affecting carpel number and ovary morphology. While ovary and fruit size were positively correlated, no relationship was found between ovule and seed size, suggesting that genes related to seed size generally act after fruit set. The collection was genotyped at the Fasciated (Fas) locus, and 13 floral traits were significantly different in fas mutants. The phenotypic variability described in this study could help breeders select for more fertile flowers and assist reproductive biologists in linking genes to flower development.

Tomato high pigment-2 (hp-2dg, hp-2, and hp-2j) mutant lines are characterized by mutations in the DE-ETIOLATED1(SlDET1; Solyc01g056340) gene. SlDET1 is responsible for encoding a nuclear protein that acts as a negative regulator involved in light signaling, repressing photomorphogenesis. These tomato mutant lines are known for increased levels of antioxidant pigments in fruits, such as flavonoids and carotenoids, compared to the wild-type fruits. In this study, CRISPR/Cas9 followed by the non-homologous end joining mechanism of repair (NHEJ), was used to target the SlDET1gene and investigate whether the effects of these mutations could mimic the effects of hp-2 mutant lines, improving the nutritional features of tomato fruits. Our results indicated that mutations generated by CRISPR/Cas9 NHEJ in the hp-2and hp-2j regions (exon 11) resulted in significant changes in the SlDET1 coding and protein sequences, causing a low survival rate of edited sprouts and regenerated plants with very compromised capacity of allelic heritability of these mutations for the following generations. However, regenerated plants containing these site-specific mutations in the SlDET1 gene showed higher levels of phytochemicals in ripe fruits. Furthermore, these edited plants also showed an upregulation of structural genes involved in the synthesis of these biocompounds. Although the SlDET1 gene could be considered an interesting target gene for the nutritional improvement of tomato fruit, our results showed that mutations within its exon 11 are quite critical and can induce severe perturbations in plant metabolism, physiology, and survival, with a compromised possibility to develop new stable edited lines.

Tomato mating systems were strongly affected by domestication events. Mutations disrupting self-incompatibility paralleled by changes retracting the stigma position (SP) within the staminal cone conferred strict autogamy and self-fertility to the cultivated forms. Although major genes affecting these changes have been identified, SP control in domesticated forms that retain a constitutive or heat-inducible noninserted SP needs elucidation. To widen the possibility of identifying SP genetic determinants, we analyzed the trait in four populations (two germplasm collections, a multiparental recombinant inbred and a biparental progeny) under different environmental conditions (normal and heat stressed). Overall, 37 markers significantly associated with the trait were identified. Several colocalizations were found, both among regions first reported in this work and among them and previously reported positions. This finding supported the reliability of the analysis. Three such regions, in the long arms of chromosomes 1, 8 and 11, were validated in an independent segregating population, and candidate genes in confidence intervals were identified among transcription factors and hormone-, stress- and cell wall-related genes. Overall, this work supported the hypothesis that the SP phenotype is controlled by different key genes in tomato, paving the way for the identification of novel players and novel mechanisms involved in the regulation of herkogamy.

The genetic diversity of tomato in Italy and the growing interest in high-quality food products highlight the importance of establishing varietal distinctiveness through molecular strategies to ensure agrifood product quality and traceability. In this study, four Italian potato-like leaf (PL) landraces were analyzed: “Spagnoletta di Formia e di Gaeta” (SPA) from southern Lazio, “Giagiù” (GIA) and “Patanara” (PTN) from Campania, and “Pomodoro di Mola” (MOL) from Apulia. These landraces were genotyped for the potato leaf gene (C), with two PL American genotypes and a non-allelic PL mutant line included as outgroups. Nagcarlang served as control. An allelism test confirmed C as determinant gene. The SCAR marker for C revealed that the Italian landraces presented determinants other than the most representative one responsible for PL. Whole-genome sequencing of SPA identified a private novel nonsense SNP variant allele, confirmed through dCAPS marker analysis. Additionally, two novel PL alleles responsible for missense variations were identified in GIA/PTN and MOL. In silico protein analysis suggested that novel C alleles could be functional determinants for the protein activity. Overall, PL mutations identified for the first time could serve as molecular tools for agrifood chain traceability, enabling early differentiation and recognition of genotypically similar varieties.

The tomato mating system was strongly affected by domestication events. Mutations disrupting self-incompatibility paralleled by changes retracting the stigma position (SP) within the staminal cone conferred strict autogamy and self-fertility to the cultivated forms. Although major genes affecting these changes have been identified, SP control in domesticated forms retaining a non-inserted or a heat-inducible SP needs elucidation. To widen the possibility of identifying SP genetic determinants, we analysed the trait in four populations (two germplasm collections, a multiparental recombinant inbred and a biparental progeny) under different environmental conditions (normal and heat stressed). Overall, 36 markers significantly associated with the trait were discovered. Several co-localizations were found, both among regions firstly reported in this work and among them and previously reported positions. This supported the reliability of the analysis. Three of such regions, in the long arms of chromosome 1, 8 and 11, were validated in an independent segregating population and candidate genes in confidence intervals were identified among transcription factors and hormone, stress and cell-wall-related genes. In conclusion, the work supported the hypothesis that the SP phenotype is controlled by different key-genes in tomato, paving the way to identifying novel players and novel mechanisms involved in the regulation of herkogamy. Highlight: The study of three germplasm collections in tomato allowed the identification new significant markers for the stigma position trait while confirmed several previously reported QTLs.

Parthenocarpy allows fruit set independently of fertilization. In parthenocarpic-prone tomato genotypes, fruit set can be achieved under pollen-limiting environmental conditions and in sterile mutants. Parthenocarpy is also regarded as a quality-related trait, when seedlessness is associated with positive fruit quality aspects. Among the different sources of genetic parthenocarpy described in tomato, the parthenocarpic fruit (pat) mutation is of particular interest because of its strong expressivity, high fruit set, and enhanced fruit quality. The complexity of the pat “syndrome” associates a strong competence for parthenocarpy with a complex floral phenotype involving stamen and ovule developmental aberrations. To understand the genetic basis of the phenotype, we mapped the pat locus within a 0.19-cM window of Chr3, comprising nine coding loci. A non-tolerated missense mutation found in the 14^th exon of Solyc03g120910, the tomato ortholog of the Arabidopsis HD-Zip III transcription factor HB15 (SlHB15), cosegregated with the pat phenotype. The role of SlHB15 in tomato reproductive development was supported by its expression in developing ovules. The link between pat and SlHB15 was validated by complementation and knock out experiments by co-suppression and CRISPR/Cas9 approaches. Comparing the phenotypes of pat and those of Arabidopsis HB15 mutants, we argued that the gene plays similar functions in species with fleshy and dry fruits, supporting a conserved mechanism of fruit set regulation in plants.

European traditional tomato varieties have been selected by farmers given their consistent performance and adaptation to local growing conditions. Here we developed a multipurpose core collection, comprising 226 accessions representative of the genotypic, phenotypic, and geographical diversity present in European traditional tomatoes, to investigate the basis of their phenotypic variation, gene×environment interactions, and stability for 33 agro-morphological traits. Comparison of the traditional varieties with a modern reference panel revealed that some traditional varieties displayed excellent agronomic performance and high trait stability, as good as or better than that of their modern counterparts. We conducted genome-wide association and genome-wide environment interaction studies and detected 141 quantitative trait loci (QTLs). Out of those, 47 QTLs were associated with the phenotype mean (meanQTLs), 41 with stability (stbQTLs), and 53 QTL-by-environment interactions (QTIs). Most QTLs displayed additive gene actions, with the exception of stbQTLs, which were mostly recessive and overdominant QTLs. Both common and specific loci controlled the phenotype mean and stability variation in traditional tomato; however, a larger proportion of specific QTLs was observed, indicating that the stability gene regulatory model is the predominant one. Developmental genes tended to map close to meanQTLs, while genes involved in stress response, hormone metabolism, and signalling were found within regions affecting stability. A total of 137 marker–trait associations for phenotypic means and stability were novel, and therefore our study enhances the understanding of the genetic basis of valuable agronomic traits and opens up a new avenue for an exploitation of the allelic diversity available within European traditional tomato germplasm.

The work analyzed the effect of two types of low-input farming conditions on the yield components and on the bioactive compounds in the pulp and peel of tomato fruits. The first low-input (LI) system involved the application of cow manure and manual weed control; in the second (LI^MI), the same system was integrated with mulching (the wood chips of eucalyptus) and intercropping (basil and cabbage plants). The study included the line 392, harboring the hp-2 gene that increases the pigments of plant and fruit; the line 446 with the atv and Aft genes which influence the content of polyphenols; and a commercial control (cv. Rio Grande). The experimental design was a split-plot where the farming system (LI and LI^MI) was allocated in the main plot and the genotype was in the sub-plot. Within the main plot, each genotype was replicated three times in three randomized blocks. Mulching and intercropping led to a differentiation in the LI^MI with respect to the LI system with higher values of the leaf greenness index (61.3 vs. 53.3 Spad units), the number of fruits (70 vs. 46), and the weight of fruits per plant (2716.6 vs. 2195.0 g). However, the LI system showed a higher content of polyphenols (+37.9%) and anthocyanins (+116.7%) in the peel and a higher content of vitamin C (+44.0%) and polyphenols (+11.1) in the pulp. The less complex LI system stimulated the plants to produce natural antioxidant systems to contrast biotic and abiotic offenders, while the introduction of mitigation elements in the LI^MI system reduced the need for protective barriers against the environmental stress. The study also revealed that low-input systems can allow for satisfactory yields, minimizing the use of off-farm resources. Growers can combine factors of sustainable agriculture with specific genotypes to maximize the production of healthier foods.