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In the post-genomic era, one of the major challenges is the identification of alleles directly responsible for phenotype variation among different genotypes within the same species. Tomato is a model crop for understanding the development and ripening of climacteric fleshy fruits, and it is also known to be an important source of health-promoting compounds. In addition, cultivated tomato germplasm shows a high phenotypic variation despite its very low genetic diversity. Toward the identification of sequence variations responsible for stress tolerance, high fruit quality and long shelf life, we re-sequenced the genomes of two traditional landraces grown in the Campania region (Southern Italy). Crovarese, belonging to the Corbarino type (COR), and Lucariello (LUC) are typically grown under low water regimes and produce highly appreciated fruits, which can be stored up to 4-8 months. We generated 65.8M and 56.4M of paired-end 30-150 bp reads with an average insert size of 380 bp (± 52bp) and 364 bp (± 49bp) for COR and LUC, respectively. A referenceguided assembly was performed using 'Heinz 1706' as a reference genome. We estimated a mean coverage depth of ~15X for COR and 13X for LUC. Comparing the genomes of COR and LUC with that of 'Heinz 1706' we found a similar distribution of SNPs (68.8% vs. 69.9%, respectively), small deletions (8.9% vs. 8.6%) and small insertions (22.1% vs. 21.3%). Through a de novo assembly of the unmapped reads we identified 29 and 36 new contigs in COR and LUC, respectively. The new contigs could be assigned to the chromosomes thanks to the use of a splitread approach. On average, the contigs inserted in COR were 654bp, whereas those inserted in LUC were 616bp. Using custom RNA-seq data, a total of 43054 and 44576 gene loci were annotated in COR and LUC, corresponding to 62369 and 65094 transcripts, respectively. Among the genes showing a similar structure in COR and LUC compared to 'Heinz 1706', we identified ~2000 and 1700 SNPs causing potentially disruptive effects on the function of 1371 and 1201 genes in COR and LUC, respectively. Interesting GO categories highly represented in genes affected by sequence changes were identified. Major variations were present in stress-responsive genes as well as in fruit quality and development-related genes. From a practical perspective, the identified SNPs and InDels are candidate polymorphisms to track DNA variations associated to key traits of economic interest.

Improving fruit quality and storability is crucial to alleviate quantitative as well as qualitative post-harvest losses that occur worldwide, and particularly in developing countries. In this study, we investigated the regulatory networks underlying tomato fruit shelf-life in the landrace Lucariello (LUC) belonging to the so-called 'da serbo' tomatoes. LUC fruits have a regular timing of ripening associated with a prolonged shelf-life of more than 150 days. The physiochemical characterization of LUC fruits is consistent with their shelf-life extension. In particular, the changes in fruit firmness and fresh weight confirmed the delay in fruit softening and withering during post-harvest. We compared by multi-omics analysis fruits at 60 and 150 days of post-harvest to those at an advanced stage of ripening, i.e. red ripe fruits at 8 days after breaker coincident with the harvest. Gene expression was quantified by RNA-seq while epigenomic analysis included the profiling of DNA methylation, small non-coding RNAs, and post-translational modifications of histones. The expression profiles of LUC fruits were compared with those from public dataset of fruits during ripening in the short shelf-life cultivar Ailsa Craig (AC). Principal Component Analysis highlighted that LUC samples at harvest do not cluster with the corresponding stage of AC fruits thereby suggesting an overall delay of over-ripening program in LUC fruits. In particular, the delay appears to be highly correlated with the downregulation of RIPENING INHIBITOR (RIN), encoding a ripening-specific MADS-box transcription factor. RIN, as well as its targets such as PECTATE LYASE (PL), a fruit softening-specific gene, and LoxB, involved in the loss of cellular membrane integrity, showed a significant downregulation during ripening in LUC with respect to AC as observed by Real Time PCR. We argue that the decreased expression of these genes in LUC is an important factor in determining its extended shelf-life, since downregulation of these genes has been shown to result in fruits that are firmer for longer than controls. In order to understand the mechanisms underlying the observed transcriptional changes, an integrated analysis is now being carried out to investigate the regulation of gene expression in relation to epigenetic modulations

Tomato (Solanum lycopersicum L.) is the model system for investigating the molecular basis of ripening in fleshy fruits. Ripening is orchestrated by a complex regulative network including multiple transcription factors, ethylene signaling pathway, and epigenome changes. It is still unclear whether fruit ripening has to be regarded as a process of senescence or as a distinct process. We investigated the biological relevance of histone post-translational modifications (HMs) H3K4me3, H3K9/K14ac, and H3K27me3 in fruit post-harvest using ChIP-Seq. H3K4me3 and H3K9/K14ac are histone marks that positively correlate with gene expression while H3K27me3 plays a repressive role. In this study, HMs were mapped in fruit pericarp of Lucariello, a landrace with long shelf-life belonging to "Pomodorino del Piennolo del Vesuvio DOP". Three different time points have been analyzed: red ripe fruit (Breaker+8) coincident with the harvest, 60 and 150 days after harvest. Based on HM profiles, the genome was partitioned into eight chromatin states, which correspond to regions marked by only one HM, co-occurrence of HMs and unmarked regions. H3K27me3-only regions were strongly associated with repetitive sequences. Chromatin states marked by H3K4me3 and/or H3K9/K14ac were found to be associated with gene bodies and promoters. Based on chromatin state changes between time points, we estimated that 12% of chromatin is dynamic during post-harvest. One-third of the changes is related to gain/loss of H3K27me3 mark. We identified differentially enriched peaks of HMs. We focused on those at +/-2kb from predicted transcription start site regions that correlate with differentially expressed genes. Data gathering and interpretation are now being carried out to identify the relative contribution of HMs to transcriptional regulation in tomato fruit post-harvest.

Tomato is a high value crop and the primary model for fleshy fruit development and ripening. Breeding priorities include increased fruit quality, shelf life and tolerance to stresses. To contribute towards this goal, we re-sequenced the genomes of Corbarino (COR) and Lucariello (LUC) landraces, which both possess the traits of plant adaptation to water deficit, prolonged fruit shelf-life and good fruit quality. Through the newly developed pipeline Reconstructor, we generated the genome sequences of COR and LUC using datasets of 65.8M and 56.4M of 30-150bp paired-end reads, respectively. New contigs including reads that could not be mapped to the tomato reference genome were assembled, and a total of 43, 054 and 44, 579 gene loci were annotated in COR and LUC. Both genomes showed novel regions with similarity to Solanum pimpinellifolium and Solanum pennellii. In addition to small deletions and insertions, 2, 000 and 1, 700 single nucleotide polymorphisms (SNPs) could exert potentially disruptive effects on 1, 371 and 1, 201 genes in COR and LUC, respectively. A detailed survey of the SNPs occurring in fruit quality, shelf life and stress tolerance related-genes identified several candidates of potential relevance. Variations in ethylene response components may concur in determining peculiar phenotypes of COR and LUC.

Quantitative and qualitative post-harvest losses occur worldwide up to 50%. It would be of great benefit to control fruit senescence thereby improving fruit quality and storability. Tomato (Solanum lycopersicum L.) is probably the best model for understanding the fleshy climacteric fruit ripening process. However, it is still unclear whether fruit ripening is a process of organ senescence or fruit senescence is a distinct process downstream the ripening. To accomplish a comprehensive investigation on the senescence-related processes, a multi-omics approach including RNA-seq for coding and non-coding RNAs, BS-seq for DNA methylation, and ChIP-seq for histone post-translational modifications (H3K4me3, H3K27me3, H3K9K14ac) was planned in tomato by using the landrace Lucariello (LUC), recognized as 'Pomodorino del Piennolo del Vesuvio', that has a shelf-life higher than 150 days. Fruit pericarps were sampled at the red ripe stage, 60 and 150 days in post-harvest (dph). Dry weight, soluble solids content and firmness decreased in LUC fruits at 150 dph by 13%, 10% and 67%, respectively, in comparison with the earliest time point. Differentially expressed genes (DEGs, n=10285) from RNA-seq included many transcription factor genes involved in fruit ripening-related processes. The occurrence of a genome-wide hypermethylation at CG and CHG sites in 150 dph fruits was concomitant with a progressive silencing of the fruit-specific DNA demethylase, SlDML2, thereby suggesting a role for the epigenome in fruit senescence. We focused on DEGs that were likely regulated by DNA methylation as well as by the abovementioned histone marks or miRNAs. In particular, more than 2000 putative miRNAs were associated with anti-correlated DEG targets in a predictive manner. Among the regions of significant H3K4me3, H3K27me3, H3K9K14ac ChIP enrichment, the peaks of read counts that were different between time points and located around the transcription start site were correlated with DEGs. Similarly, the regions that significantly gained/lost DNA methylation across time points (Differentially Methylated Regions) were analysed for the association with DEGs. Based on the identified DEGs, co-expression analysis is currently underway to find activator and/or repressor candidates in the regulatory pathways underlying tomato fruit senescence. Furthermore, all the data are being reconciled in a web-based platform.

Tomato (Solanum lycopersicum L.) is a high value horticultural crop and an important dietary source of nutrients, vitamins and antioxidants. The cultivation is often subjected to drought, salinity and N-limited conditions. Thus, understanding the responses of tomato to salt and low N conditions will be instrumental to improve yields in stress-prone environments. In this study, physiological and molecular responses of three Italian landraces (TRPO0040, TRPA0130 and TRPO0670) to salt stress and low nitrate, alone or in combination were analysed. The experimental set up allowed four different treatments named: Control (13.5 mM NO3- - 0 mM NaCl), Salt stress (13.5 mM NO3- - 80 mM NaCl), N stress (3.4 mM NO3-- 0 mM NaCl) and Combined stress (3.4 mM NO3- - 80 mM NaCl). The treatments had different effects on plants: low N caused chlorosis, while salt stress inhibited growth causing loss of basal leaves. Leaf relative water content was affected by both salt and low nitrate, depending on the genotype, while proline content was dramatically increased by salt stress but not under combined stress. Growth parameters and fruit yield were reduced by single stress condition, and the highest reduction was observed in the combined stress. TRPO0040 genotype showed high Nitrogen Use Efficiency and Nitrogen Utilization Efficiency under salt, indicating that nitrogen allocation to fruits was not affected by this stress. We evaluated the impact of single and combined stresses on tomato transcriptome by performing RNAseq on roots and leaves of TRPO0040 after long-term exposure to the treatments. In leaves, a more extensive transcriptome reorganization was observed in response to N stress, while salt induced a significant variation in gene expression of a handful of genes. Parallel to the proline levels, expression of P5CS gene, encoding the rate limiting proline biosynthetic enzyme, was induced by salt, while the catabolic proline dehydrogenase was suppressed. Similarly, several genes encoding nitrate transporters were induced in N stress, including NRT2;2 and others. By contrast, in roots several thousands of differentially expressed genes (DEGs) DEGs in salt stress and only few hundreds in N stress were identified. Consistent with results gathered in Arabidopsis, SLAH1, a gene encoding a root specific anion transporter involved in the long distance transport of MOLECULAR MECHANISMS UNDERLYING MORPHO- PHYSIOLOGICAL ADAPTATION TO COMBINED SALT/LOW NUTRIENT STRESS IN TOMATO chloride ions, was upregulated in roots in salt stress condition, while a NRT2;4 like and AMT1 were induced by low nitrate. In the combined stress, a more extensive overlap of the DEGs was observed with the N stress rather than the salt stress for both roots and leaves. This indicates that nutrient availability may have a higher and longer- term impact on gene expression, compared to salt stress, which may induce rapid and transient responses, attenuated, particularly in leaves, when plant adaptation occurs. Altogether, we show that different cultivation regimes affect metabolic and transcriptomic profiles as well as growth in tomato, identifying a set of physiological and molecular targets specifically influenced by single or combined stress.

Tuta absoluta Meyrick (Lepidoptera: Gelechiidae) is native to South America and has rapidly spread in various countries, becoming one of the major threats to tomato crop (Solanum lycopersicum L.). Its range is constantly expanding, spacing up to the North-Western Himalayan Region of India and in the sub-Saharan Africa area. Although this species has a tremendous potential to develop resistance against many insecticides, its control still relies largely on molecules such as organophosphates, pyrethroids, spinosad and diamides. Sustainable control, either based on the use of predators (i.e mirid bugs) and eggs parasitoids (i.e. Trichogrammatidae) or mating disruption (also combined), has been successful only in specific conditions. In this context, the exploitation of resistance traits from wild relative species could offer another sustainable alternative to synthetic insecticides. It is noteworthy that crop wild relatives harbour an abundant supply of resistance genes for biotic and abiotic stresses. We studied two wild tomato backgrounds, Solanum pennellii (acc. LA0716) and Solanum habrochaites (acc. LA1777), using the corresponding populations of introgression lines (ILs and ILSHs) obtained by crossing the processing tomato inbred varieties M82 and E6203 to the wild accessions LA0716 and LA1777, respectively. These lines are expected to exert defence mechanisms able to hamper the attraction and the larval feeding of T. absoluta. Pest resistance of these wild tomato accessions has been related to naturally occurring allelochemicals present in leaf glandular trichomes such as acyl sugars, 2-tridecanone, or sesquiterpenes, especially zingiberene. We have selected a panel of five lines of both IL populations, and have carried out bioassays to compare their effect on T. absoluta to identify genes putatively involved in resistance to this invasive pest. The number of eggs laid over time unit was used as a measure of adult attraction, while antixenosis/antibiosis was measured by recording survival, development time and pupal weight of feeding larvae on tomato leaves. Our work showed that several ILs exhibit an intermediate resistance level, prompting further studies to characterize the specific gene/s involved. Nonetheless, breeding programmes could benefit from these preliminary results.

A better understanding of regulatory networks underlying fruit senescence would greatly impact on fruit quality and storability. Tomato (Solanum lycopersicum L.) is currently the best model for studying ripening process in climacteric fleshy fruit. However, the boundary between fruit ripening and senescence is still unclear. The Italian landrace Lucariello (LUC), recognized as 'Pomodorino del Piennolo del Vesuvio', is characterized by a long shelf-life reaching more than 150 days after harvest, thus it represents an excellent model to investigate the relationship between ripening and senescence and a genetic resource to transfer its long shelflife to other cultivars. For a full characterization of Lucariello, a multi-omics approach was followed, in which the genome was resequenced, assembled and annotated. In addition, coding and non-coding RNAs, DNA methylation, and histone post-translational modifications (H3K4me3, H3K27me3, H3K9K14ac) were analysed in fruit pericarps at the red ripe stage, 60 and 150 days in post-harvest (dph). More than 10,000 genes were found to be differentially expressed across the time points and many were related to transcription factors and to fruit ripening. About 2,000 putative miRNAs were predicted as anti-correlated to DEG targets. Differentially enriched regions for H3K4me3, H3K27me3, H3K9K14ac and differentially methylated regions were identified around the transcription start site and correlated with gene expression. An integrated analysis is now being carried out to find activator and/or repressor candidates regulating senescence process in tomato fruits. All the genomic and epigenomic datasets are gathered into a dedicated and user-friendly web-based platform.

Increased interest towards traditional tomato varieties is fuelled by the need to rescue desirable organoleptic traits and to improve the quality of fresh and processed tomatoes in the market. In addition, the phenotypic and genetic variation preserved in tomato landraces represents a means to understand the genetic basis of traits related to health and organoleptic aspects and to breed them in modern varieties. In the frame of Horizon 2020, the TRADITOM project "Traditional tomato varieties and cultural practices: a case for agricultural diversification with impact on food security and health of European population" (http://traditom.eu/) aims to the collection, characterization and valorisation of European traditional tomato germplasm. The project has the long-term objective to understand biochemical and molecular factors underlying the typicality of traditional tomatoes, in the perspective to genetically improve them without losing quality traits and to eventually breed quality alleles into modern genetic backgrounds. As a part of the whole project collection of over 1500 accessions, this presentation describes the characterization of the main collections preserved in Italy, listing a total of over 430 samples. In two different phenotypic trials, the material has been scored for 18 qualitative variables and measured for further eight plant and fruit traits. Image analysis was conducted with the Tomato Analyzer software allowing for 15 morphometric and nine colorimetric parameters to be detected. A subset of the collection was subjected to metabolic profiling. The accessions showed a wide variability for most scored traits, including, for example, a 90-fold variation for fruit weight, 8-fold for locule number and >4-fold for the soluble solid and carotenoid content. Correlation and ANOVA statistics indicated distinctive differences among groups of accessions belonging to recognized typologies. The classification based on morphology was compared with genotypic data collected through 'genotyping by sequencing'.

Chamomile (Matricaria recutita), a member of the Asteraceae family, is a good source of health-related compounds such as sesquiterpenes, coumarins, polyacetylenes and flavonoids including apigenin. This flavone exhibits interesting in vitro and in vivo pharmacological activities as anti-oxidant, anti-inflammatory, and anti-cancer properties. Moreover, recently it has been established that apigenin significantly decreased the blood levels of total and low-density lipoprotein cholesterol in mice. Apigenin is synthesized in planta by the phenylpropanoid pathway only in very limited amounts. In this study we investigated the apigenin pathway in chamomile in order to pave the way to increase the production of this valuable compound by metabolic engineering approches. For this purpose, the full-length cDNA of flavone synthase gene (FNS), encoding the key enzyme of the apigenin pathway, was isolated from M. recutita using a similarity-based cloning strategy. Nested PCRs with degenerate primer sets, designed to match the highly conserved regions of other FNS genes, were carried out to amplify the core fragment of 510 bp. PCR-RACE was applied for amplification of 3' and 5'-ends. Comparative analysis showed that chamomile FNS sequence was closely related to other flavone synthases, showing a high degree of amino acid identity with flavone synthase II of Cynara cardunculus var. scolymus. Expression pattern of the M. recutita flavone synthase gene was achieved by Real-Time in roots, stems, leaves and flowers. In addition, a method of stable genetic transformation by Agrobacterium tumefaciens was set up in M. recutita using the LBA4404 strain harboring a binary vector with marker gene neomycin phosphotransferase (NPTII) and reporter gene ?-glucuronidase (GUS). This protocol could become a useful tool for metabolic engineering approches in chamomile.

Tomato (Solanum lycopersicum L.) is an important horticultural crop and a significant source of antioxidants in our diet. Yields suffer losses due to environmental stresses, including drought and salinity, which are among the most significant challenges for the future of global food production. Reduction of fertilizer inputs, including nitrate is also a priority in plant research, and it is aimed at minimizing pollution caused by agricultural activities, while obtaining fruits of high quality and long storability. In this study, we analysed vegetative and molecular responses of three tomato landraces (TRPO0040, TRPA0130 and TRPO0670) challenged with salt stress and low nitrate, alone or in combination, and their effects on fruit yield, quality and storability. The experiment was setup in a closed soilless system, in greenhouse, using four different conditions: N1S0: 13.5 mM NO3- - 0 mM NaCl; N1S1: 13.5 mM NO3- - 80 mM NaCl; N0S0: 3.4 mM NO3-- 0 mM NaCl; N0S1: 3.4 mM NO3- - 80 mM NaCl. Several parameters were monitored during the vegetative phase, including stomatal conductance, leaf relative water and proline content, expression of selected ion transporter genes. Relative water content was affected by both salt and nitrate concentration, while proline content was dramatically increased by salt stress in high nitrate condition only. Growth parameters as well as fruit yield were reduced in the presence of stress, with the highest reduction observed in plants exposed to the combined stress. Highest yields were obtained in N1S0, however fruits harvested from low nutrient-treated plants were virtually all marketable, regardless the presence of NaCl. Soluble solids content was highly affected by salt, as expected. The cultivation regime also affected fruit shelf life of the three genotypes over the 90-day period monitored. High salt in the nutrient solution improved fruit shelf life regardless of genotype and nitrate concentration. In the absence of NaCl, NO3- supply in the nutrient solution did not affect fruit shelf life in TRPO0670 and TRPA0130, while TRPO0040 fruits showed an extended shelf life at low nitrate compared to high nitrate supply. Fruit firmness and weight declined in all genotypes and treatments during post-harvest; hence, they were not ?? RUGGIERO A.*, BATELLI G.*, VENEZIA A. ??NURCATO R.*, COSTA A.*, ?? discriminating parameters between treatments. Nitrogen Use Efficiency (NUE) evaluation in all treatments and landraces revealed that TRPO0040 maintained high NUE under salt stress, indicating that N allocation to fruits was not inhibited by salt in this genotype. Altogether, we have verified that different cultivation regimes affect fruit shelf life and quality in tomato, and identified a set of vegetative parameters specifically influenced by single or combined stress. In addition, RNAseq and qRT-PCRs analyses are being pursued in order to get further insights into the molecular basis of fruit shelf life under different cultivation regimes.

Epigenetics has been proven to be involved in the regulation of tomato fruit ripening, but a comprehensive investigation on post-harvest related processes is still lacking. By applying next generation sequencing technologies, the EPITOM Consortium is performing integrative analyses including coding and non-coding RNAs, DNA methylation, and histone post-translational modifications in long shelf-life traditional variety Lucariello, recognized as 'Pomodorino del Piennolo del Vesuvio PDOP'. Fruit pericarps have been sampled at the red ripe stage and at two and five months in post-harvest. Currently, the data of high throughput sequencing of mRNA, small RNAs (sRNA), and long non-coding RNAs (lncRNA) are available. RNA-sequencing (RNA-Seq) analysis allowed the identification of several new genes in comparison to the current tomato genome annotation (ITAG2.4, SL2.50). In addition, a large number of alternative transcript variants were identified at the post-harvest stages compared to the red ripe one. Gene ontology (GO) enrichment analysis of all differentially expressed genes highlighted nine over-represented GO terms involved in epigenetic mechanisms, thereby suggesting a pivotal role of the epigenome during tomato post-harvest. Putative miRNAs were also predicted to target differentially expressed mRNAs involved in fruit ripening-related processes. In addition, a number of miRNA precursors showed upstream binding sites for stress related transcription factors. LncRNAs, isolated from RNA-seq experiment selecting ORFs longer than 200 nt are under evaluation to identify novel lncRNAs and to investigate their role in the regulatory mechanisms of tomato fruit ripening during post-harvest. All the data are being reconciled in a web-based platform to allow multilevel integrated analyses of the available collections.

— Chromosome number and pollen stainability were evaluated in Lilium L. species (L. formosanum, L. miryophillum, L. regale), and in cultivars coming from Asiatic (Elite, Pollyanna, Vivaldi) and Oriental hybrid group (Cascade, Casablanca, Galilei). All the genotypes are diploid (2n=2x=24) except cv. Elite which results triploid (2n=3x=36). The pollen stainability ranged from 90 to 98% among diploids and from 80 to 90% in the triploid which exhibited pollen grains of different size, as well. A detailed analysis of microsporogenesis was carried out in the triploid cultivar. The chromosome associations at diakinesis were either trivalents or bivalents and univalents. Anaphases I and II exhibited lagging chromosomes and unequal distribution of chromosomes at the two poles. Single and double bridges plus acentric fragments formed possibly due to a paracentric inversion. Meiotic nuclear restitution mechanisms occurred following the failure of the reductional wall or, alternatively, of the equational wall. The analysis of sporads evidenced tetrads as well as dyads, triads and rare monads and poliads.

Fourteen species of Aster were characterized for morphological traits of commercial interest, chromosome number and genetic diversity by AFLP markers to exploit this material not only for breeding purposes but also as source of pharmaceuticals. The variation observed among the Aster species for five morphological traits was summarized by means of discriminant analysis. Three significant canonical variables, accounting for about 96% of total variance, were mainly correlated with capitulum diameter, number of ligulae and leaf length. The morphological variation appeared related to ploidy level of the species. A diploid chromosome number ranging from 10 to 18 was evident in seven species whereas the other species are polyploids with variable chromosome numbers up to 52. The pollen production and stainability were quite variable among Aster species. Furthermore, seven species produced big pollen grains besides normally-sized ones, at a frequency ranging from 1 to 12%, possibly due to 2n pollen production. The possibility that evolution of Aster genus could be driven by 2n gametes and sexual polyploidization is discussed. Implications of 2n gametes for Aster breeding are also mentioned. AFLP analysis of the fourteen Aster species based on six primer combinations revealed a total number of 421 polymorphic amplified DNA fragments. The genetic similarities estimated between the Aster species, based on both shared and unique amplification products ranged from 0.335 to 0.604 across all species and revealed a geographically based clustering within the genus. The highest similarity was detected between A. alpinus and A. amellus with Eurasian origin.

Lupeol is a pentacyclic triterpene found in fruit, vegetable as well as in several medicinal plants. This compound shows interesting pharmacological activities including anti-inflammatory, anti-tumor, anti-mutagenic and anti-malaria properties. Moreover, it has been demonstrated that dietary supplementation of lupeol prevents cancer, coronary, and hepatic diseases. Lupeol biosynthesis occurs in the citosol through the mevalonate (MVA) pathway that leads to the formation of 2,3-oxidosqualene, which is then cyclized in lupeol by lupeol synthase. In this work, the full-length cDNA of a lupeol synthase, designated CaLUP, was isolated from pepper (Capsicum annum L.). The CaLUP open reading frame consisting of 2271 bp was predicted to encode a protein of 756 amino acid residues. The comparative and phylogenetic analysis of CaLUP showed that this protein is closely related to other plant lupeol synthases, sharing 66-90% identity with their amino acid sequences. In particular, a high degree of identity (80%) was observed with lupeol synthase of Solanum tuberosum. Genotype-and tissue-specific expression patterns of the CaLUP were determined by quantitative real-time PCR analysis (qRT-PCR). In silico promoter analysis of CaLUP identified distinct cis-regulatory elements; some of these were selected to investigate their function in elicitor-induced plant response. For functional analysis, the cDNA clone containing the full-length ORF of the putative lupeol synthase is being cloned into the expression vector pYES2, and will be transformed into the yeast mutant strain GIL77.

Pepper (Capsicum annum L.), a member of the Solanaceae family, is an economically important crop, being used worldwide as a vegetable, spice and food additive. Pepper fruits show a remarkable range of morphological variation, particularly in terms of shape and size. These traits are important factors determining yield, quality and consumer acceptability for many crops. In tomato, the major QTL affecting fruit shape and size have been identified, and for some of them, including ovate, sun, fasciated (fas) and fw3.2, the underlying genes have been cloned (OVATE, SUN, FAS and KLUH, respectively). In pepper, among the several fruit shape and size QTL identified so far, only the OVATE gene has been cloned. In this study, using comparative analysis, the putative ORFs of SUN, FAS and KLUH genes have been identified in pepper (namely CaSUN, CaFAS, and CaKLUH). CaFAS and CaSUN, consisting of 540 bp and 1248 bp, were predicted to encode proteins of 179 and 415 amino acid residues, respectively. The deduced amino acid sequences of CaFAS and CaSUN showed the YABBY and IQ67 domains, respectively. In addition, CaKLUH ORF consisting of 1551 bp was also isolated and cloned. Amino acid sequence alignments revealed that CaKLUH shares high sequence identity with cytochrome P450 78A4-like of Solanum lycopersicum (XP_004236064; 100%). In order to assess the structure of CaKLUH gene, we also cloned the genomic sequence. Alignment between the genomic and cDNA sequences revealed that the CaKLUH gene spanned 2.4 Kb in length and was organized into 2 exons and 1 intron. The expression patterns of the three putative genes were determined by Real-Time PCR in different organs and development stages of four pepper cultivars characterized by different fruit shapes and sizes. For functional analysis, development of TRV based VIGS vectors is in progress.

Pepper (Capsicum annuum L.) is an economically and nutritionally important crop belonging to Solanaceae family. The recent availability of the pepper genome sequence demands the development of more efficient tools for gene functional analysis. However, pepper is a recalcitrant species for shoot regeneration, and although a few transient and stable transformation protocols have been published, the study of pepper gene function is still a difficult task. In addition, pepper transformation efficiency varies among different cultivars. In this study, a transient genetic transformation protocol has been optimized for Capsicum annuum cv. "Quadrato d'Asti" using a TRV-based VIGS vector and phytoene desaturase (PDS) as reporter gene. Leaves and seedlings have been infiltrated with Agrobacterium tumefaciens strain GV3101 carrying TRV plasmids, which are a bipartite vector system. Photobleaching appeared after approximately eight days and three weeks on detached leaves and seedlings, respectively. It was observed that infiltrated plants showed photobleaching even four months after infiltration and produced photobleached fruits. Molecular analysis showed that CaPDS transcripts were dramatically reduced in the silenced leaf. In order to reduce the time of symptoms appearance, agroinfiltration has also been tested on flowers, inflorescences and fruits at different developmental stages. As concerns stable genetic transformation, an efficient protocol for high-frequency regeneration of cv. "Quadrato d'Asti" has been set up, and A. tumefaciens-mediated transformation using the strain GV3101 is in progress. These transformation methods will be used for functional analysis of genes involved in merceological (fruit shape and size) and technological (cuticle thickness and composition) quality of pepper fruits.

The tomato fruit is an ideal and well-studied model system for investigation of fruit development, flavor and nutrition. Exceptional variation in these traits is found within germplasm of the tomato clade and numerous genetic, molecular and germplasm resources enable efficient research in the tomato system. Populations of marker-defined introgression lines (ILs) originating from interspecific crosses represent powerful tools to harness the genetic potential stored in unadapted germplasm for both fundamental biology studies and to improve the agricultural performance of modern crop varieties. Recent developments of 'omics' platforms have further enhanced the power of such congenic and permanent resources. In order to gain further insights into the networks regulating key fruit metabolic pathways underlying flavor and nutritional quality, we have undertaken a systems approach that leverages natural variation stored in a new set of COSII-anchored Solanum habrochaites (LA1777) ILs in the genetic background of the S. lycopersicum processing tomato cv. E6203. Towards this end, we have performed RNA-seq analysis from red ripe fruit pericarps of 30 S. habrochaites ILs. Single nucleotide polymorphisms (SNPs) and indel profiles were identified for each IL based on RNA-Seq data. This high-density genotyping allows precise definition of the boundaries of each IL facilitating use of this resource for breeding of nutrient, flavor in addition to any other trait of interest. In several cases, the high resolution of introgression definition achieved through RNA-seq SNP analysis allowed the identification of small additional S. habrochaites introgressions missed in lower resolution genotyping studies based on RFLP or PCR markers. Replication of RNA-seq analysis of these and additional lines is under way and the status of this developing expression resource will be presented.

Organization and rearrangements of microtubular cytoskeleton (MTs) during microsporogenesis are determined in 'historical' potato genotypes producing 2n pollen following different meiotic nuclear restitution mechanisms ('parallel spindles', 'fused spindles', 'premature cytokinesis'). The scope of the investigation is to assess the abnormalities in MT cytoskeleton and their relationships to 2n pollen formation. The genotypes of Solanum used for MT immunofluorescence in this study are diploid interspecific hybrids (2n=2x=24) coming from Wisconsin (USA) and Wageningen (NL) Universities. Based on NIT analysis, a reinterpretation of the restitution mechanisms following spindle abnormalities was performed in this study. The fusion of the nuclei at prophase II occurs through the perinuclear MTs in the genotype with 'fused spindles'. In genotypes with misoriented spindles, the 'non-spindle' cytoskeletal arrays, occurring at telophase II, are clue in determining the meiotic products such as dyads and triads. A particular NIT array could be crucially involved in meiotic nuclear restitutions both at telophase I and II: the interzonal MTs. It is discussed which is the relationship between this array and the spindle orientation at meta-anaphase II. It is hypothesized that the fusion of non-sister nuclei at each pole depends on the lack of secondary interzonal MTs and on the short distance between nuclei. In the genotype with premature cytokinesis, effectively, a premature phragmoplast occurs at the end of meiosis I. Furthermore, two genotypes are desynaptic with a high degree of male fertility restored by the nuclear restitutions. However, no clear relationship was found between univalents and restitution mechanisms.

The aim of this work is the evaluation for chipping and resistance to tuber soft rot (Erwinia carotovora subsp. carotovora) of tetraploid potato clones with germplasm from S. chacoense and S. phureja obtained via 2n eggs through 2x-4x crosses. Cytological mechanisms were investigated in the diploid hybrid 2n-egg producer used as parent in the unilateral sexual polyploidization scheme; the omission of the second meiotic division and cytokinesis failure at the end of meiosis were identified with a genetic consequence equivalent to SDR mechanism. Tuber marketable yield, specific gravity and chip-processing ability evaluated at harvest and after cold storage with and without reconditioning were higher in some clones as compared to the S. tuberosum tetraploid parent. Tuber soft rot resistance which was present in the used accession of S. chacoense was transferred at 4x level. After inoculation with Erwinia carotovora subsp. carotovora and estimation of the rotted area, one clone proved to be resistant and three partially resistant. The genetic value of exotic germplasm and sexual polyploidization strategy are discussed in relation to potato improvement.